issn 2249-4812 vol 1 no.2 oct 2011 for web...jcaesok the journal of conservative and endodontic...

ISSN 2249-4812

JCAESOKThe Journal of Conservative and

Endodontic Society of KeralaOctober 2011 | Vol. 1 - Issue No. 2

ISSN 2249 –4812

Editor in ChiefDr. Ganesh C.

Associate editorsDr. Rajesh PillaiDr. Jothish Ravi

Business ManagerDr Robin Theruvil

Editorial Advisory boardDr. Ravi Varma

Dr. N.O VargheseDr. Kunjamma Thomas

Dr. Baby JamesDr. Radhakrishnan Nair

Dr. V I PaulDr. Mohammed Sagir

Dr. Romel JosephDr. Gene Jacob

Dr. Josey MathewDr. Mohammed Sadique

Dr. Pramod kumarDr. Satheesh kumar

Dr. Thajuraj

Editorial Review panelDr. Beena Rani Goel

Dr. C.V Pradeep.Dr. Ignatius RexDr. DevadathanDr Shiju Cherian

Dr Prashant Dhanpal T.Dr. Pratap M.S

Dr. Mathew VargheseDr. George Jacob

Editorial Office“Swagat”, T. C 2/3601,

Kesavadasapuram, Trivandrum -695004Ph- 09446557487, 09349457487E-mail: [email protected]

www.caesok.org

Contents

JCAESOK • October 2011, Vol. 1, No. 2 •

JCAESOK

Designing and PrintingSuman Graphics, Trivandrum

[email protected]

For Private circulation only

ISSN 2249 –4812

Invited Review

Endodontic biofilms-Their formation and eradication: A review 54Kundabala Mala

Original Scientific Articles

Correlation between vitamin c level and dental caries– A clinical study 59

Mithra N. Hegde, Nidarsh D. Hegde, Sucheta Shetty, Vinod R. Jathanna

Effect of additional acid etching on resin tag formation with self-etching adhesives - A scanning electron microscopy study 62

Sunil Earaly, Mithra N. Hegde, Priyadarshini Hegde

Comparative evaluation of odontoblastic activity following directpulp capping with two bonding agents and calcium hydroxide inhumans-A histological analysis 67

Sreeja Sreedhar, Ramesh Kumar M.

The efficacy of pre-operative oral medication of Ibuprofen andTenoxicam on the success of inferior alveolar nerve block in patientswith irreversible pulpitis: a double-blind, randomized controlledclinical trial 74

Sulthan Ibrahim Raja Khan, Ramachandran Anupama, Mohanavelu

Deepalakshmi, Kumarappan Senthil Kumar

Multiple irradiation of enamel surface in conjunction withacidulated phosphate fluoride (APF) gel, fluoride enhancedhydroxyapatite gel (Remin Pro) and propolis-A SEM evaluation 77

Mathew Varghese K, Ajit George Mohan

Comparative evaluation of the shear bond strength of conventionaltotal etch adhesive with simplified bonding systems 81

Shamina Bawa, Mithra N. Hegde

Case reports

Post healing complication of horizontal fracture : A case report 84Swathi Amin, Prathap M.S., Harish Shetty

Revascularization of an immature non-vital permanent tooth-A case report. 87

Eva.C.Das

Review

Guided tissue remineralization: A novel method for toothremineralization 90

Sapna C M, Baby James, Rakesh, Sreedevi N B


50

Conservative and Endodontic Society of Kerala

(CAESOK)

President 1st Vice-President 2nd Vice-President

Dr.Baby James Dr.Mohammed Sagir Dr.Devadathan

President Elect Imm. Past President Secretary Joint Secretary

Dr. Radhakrishnan Nair Dr.Paul V.I Dr. Prasanth Dhanpal Dr.George Jacob

Treasurer Editor

Dr.Romel Joseph Dr.Ganesh C.

OFFICE BEARERS

Dr.Gene Jacob Dr.Pradeep C V

Dr.Josey Mathew Dr.Pramod Kumar AV

Dr.Kunjamma Thomas Dr.Prathap MS

Dr.Mathew Varghese Dr.Rajesh Pillai

Dr.Mohammed Sadique Dr.Robin Theruvil

Dr.Satheesh Kumar Dr.Thajuraj PK

Dr.Varghese NO

Executive Committee Members

The CAESOK came into being as an organization on the

28th September 2002. It was the like minded discussions

among a few Endodontists like Dr.Shiju Cherian, Dr.John

Joseph, Dr.A.K.George, Dr.Rajkrishnan and

Dr.N.O.Varghese during the dental conferences and

meetings that led to the idea of forming a society of

Endodontists from Kerala to share their views and

aspirations. These informal chats were followed up with

numerous telephonic discussions. With the sincere

support of Dr.C.V.Pradeep, Dr.V.I.Paul, Dr.Gene Jacob and

Dr.Sivapriyan, the foundations were laid, with the first

meeting of about 60-70 Endodontists from various parts of

Kerala taking place at Hotel Renaissance, Ernakulam on

3rd February 2002.

The name of the society, CAESOK, was suggested by

Dr.N.O.Varghese and the emblem for the society was

designed in the artistic hands of Dr.Ajith Thomas.

With Dr.Kunjamma Thomas and Dr.Baby James joining

the aforementioned team of dentists, these pioneers

formed the group of office bearers at the official inauguration

of the society by the Hon.Justice M.Ramachandran on the

28th September 2002 held at Riviera Suites, Kochi.

Since its inception the society holds a midterm

conference and an annual conference and family meet every

year to further strengthen the bond of friendship and

fellowship as individuals and as a family.


51

As the second edition of JCAESOK goes to print, it is

testimonial to the commitment of the endodontists of

the state towards the science. I am proud of the way

our editors have put together this edition,a rich

reserve of scientific information presented in an

esthetic setting. Their efforts are indeed laudable. In

fact every endeavor that caesok sets forth on, has a

habit of turning out commendable. Be it the academic

front or social enrichment,with conventions and

conferences ranging from interactive sessions with the

intellectual think-tank, upto the mind-blowing

overseas experience of Thailand. The one common

fiber in the success of every event is the collective

effort of our members. We think as one, work as one

and the results are for all to see and rejoice.

With the year drawing to an end, festivity is in the air

and JCAESOK second edition is one more reason for us

to get together and celebrate

Jai CAESOK!

Dr. Baby JamesPresident, CAESOK

President’s message


52

I am much delighted and pleased to write amessage in the second edition of our prestigiousjournal. The maiden issue of JCAESOK was ahuge success and got appreciation from allquarters. Acclaimed academicians, renownedclinicians and inquisitive post graduate studentsof Conservative Dentistry and Endodontics wereall appreciative of the standard of the journal.Very few specialty journals stand upto this levelin the first volume itself. My congratulations tothe editorial team for this achievement. I wouldlike to place my sincere appreciation to theEditor Dr Ganesh and his team for tirelesslyworking to make sure that the second issue isin the member's hands in time and keeping upthe standard of publication.

Improvements in techniques, instrumentationand material science have lead to greaterinterest in restorative dentistry and endodonticsin the last few years. Greater acceptance andpredictability of restorative and endodonticprocedure and need to conserve the naturaldentition among the general public has positivelyimpacted our profession. Research andpublication will aid in propagation of knowledgeand we lead by example. Our specialty journalis presently reaching members and institutionsacross the country.

Many of the dental institution in our state arein the process of starting post graduate coursein conservative dentistry and endodontics. Thisjournal would be of great help for the post

graduate students. At this juncture I wouldrequest all, members and students to take upresearch and make it a point to publish atleast1 original article per year.

We neither lack infrastructure for research norare we in a dearth of ideas for improvisation.Of the two things that may hamper our spiritfor research, one would be inadequate fundingfrom institutions, Governments or quasigovernment organization. As an organization forspecialty dental professional, we should try torepresent these ideas to the concernedauthorities and obtain grants for research inour specialty. The second thing which comes inthe way of research and publication is lack ofmotivation by professionals. It should never bea statutory need but an obligation towards theprofession, which is well respected among otherdental professionals across the world. Pleasetry to give back something to the profession,which have given you so much

With this few words, I once again urge my fellowmembers and students to contribute towardsresearch and contribute towards success of ourspecialty journal.

I love to be cared by you and I love to care foryou.

Yours sincerely.

Dr. Prasanth DhanapalHon. Secretary, CAESOK

Secretarys message


53

Editorialstandard of the scientific content.The

methodology of selection and processing

plays the most important role in ensuring

the academic standard of the Journal. We

at JCAESOK are trying to ensure the quality

of the articles in the journal. To further

improve, it is imperative for the authors to

follow the guide lines for the submission of

the articles. JCAESOK is still in its infancy

and there is a lot more work to be done to

stream line the submission and reviewing

process to make it an efficient, hassle free

and practical system. We have started the

project and need your support and

encouragement to go all the way and make

it a good scientific journal.

Dr.Ganesh C.

Editor in Chief

Professor,Dept. of Conservative Dentistry

and Endodontics,Sri Sankara Dental College,

Varkala, Trivandrum.

“There are two mistakes one can makealong the road to truth…not going all theway and not starting”.

- Buddha

The first issue of JCAESOK was released at

the PG convention of CAESOK, 2011 at

Kozhikode, Kerala. It was well received by

all and the interest shown by the seniors

and colleagues in the field was very

encouraging and has powered us to the

second issue. The valuable input given is

well appreciated and we have tried to

incorporate it in this issue.

The new DCI guideline which includes all

the authors in an original research paper in

the point system is a welcome move. A good

work of research is the result of many minds

and therefore it seems prudent to include

all the contributors in the credit points

system.

The authenticity of a journal lies in the


54

Endodontic biofilms - Their formation and eradication: A Review* Kundabala Mala

Introduction

Microoraganisms in the normal microbiota participatein many beneficial physiological functions of the oral cavity.In case the physical and biochemical conditions of thesetissues change, commensals can convert into opportunisticpathogens and produce disease especially if the organismscan gain access to the tissues in the pulp cavity andperiradicular area. Microbes are the most essential agentsfor the development of primary periredicular diseases andare most essential factors responsible for the failure ofendodontic therapy. Hence the goal of primary endodontictherapy as well as retreatment should be to eradicate themicrobes from the root canal system.(1)

It is not possible to eradicate the microbes completelyfrom the root canal system for following reasons

1. The complex anatomy of the root canal system2. The large number and diverse array of highly virulent

pathogens3. The diffuse localization of endodontic infections4. The relative antimicrobial resistance of microbes

which are continuously undergoing mutations to adapt toany situation.

5. Bacterial penetration and persistence in dentinaltubules

6. Action of dentin in reducing activities of antisepticagents

7. Biofilm formation inside the root canal system.(2-5)Several microbial species which are prevalent on most

wet surfaces have tendency to attach on radicular dentinsurface. They create an extraordinary microenvironment andform dense bacterial biofilms.(6) They can cause a persistentinfections(7,8). The biofilm mode of growth seems to beadvantageous for microorganisms as they form threedimensional structured communities with fluid channelsfor transport of substrate, waste products and signalmolecules.(9)

DEFINITION OF BIOFILM: Biofilms are defined aspolysaccharide matrix enclosed sessile or pedunculatedmicrobial population composed of tower or mushroomshaped micro colonies, containing cells irreversibly adherentto each other and /or to substratum or interfaces.(10) Theyare thin layered condensations of microorganisms whichcan include bacteria, fungi and protozoa.(11)

They are heterogeneous arrangement of microbial cellson a solid surface. Even though there is variation in thecomposition and activities of the colonizing microorganisms,the biofilm formation follows the same pattern ofdevelopmental stages.

* Associate Dean, MCODS, Mangalore,Professor, Department of Conservative Dentistry & Endodontics,Manipal college of Dental Sciences, Mangalore Light House Hill Road, Mangalore,Karnataka-575001; Email:[email protected]

Formation of biofilm

Biofilm formation is a step-wise procedure its formationoccurs in the presence of microorganisms, fluid and solidsurface. Biofilm is considered as community as it possessesfollowing criteria:

Autopoiesis: ability to self- recognizeHaemostasis: resist environmental changesSynergy: Stronger in groups than in isolationCommunality: respond to environmental changes in as a

team than as single.(12)

Characteristics of biofilm

1. Bacteria present in biofilm are protected from theenvironmental threats

2. Traps nutrients and creates metabolic cooperativitybetween resident cells of same and/or different species

3. Display organized compartmentalization for thesurvival of different kinds of bacteria

4. Creates an environment to communicate and exchangesgenetic materials with each other to acquire new traits.(13)

The organisms in biofilm differ in their character fromplanktonic bacteria. Microorganisms in biofilm require asolid surface and flowing nutritious fluid for the survivalduring starvation period. Periapical biofilms aroundrefractory teeth are independent of solid surface of the roottip. The sessile bacterial cells differ from their planktoniccounterpart by following properties:(14,15,16,17)

The phases for microbial community to developa biofilm and colonize the environment

Step 1: Deposition of conditioning film by adsorption ofinorganic and organic molecules to the substratum such assolid surface. (18,19,20,21.)

Step 2: Adhesion of planktonic microbial cells to theconditioning layer and colonization of microbes .Initial bondbetween the bacteria and substrate is weak slowly over aperiod they grow stronger with irreversible polysaccharideadhesion and this takes place in 3 phases,

Phase 1: Transport of the microbe to substrate surfaceand its attachment. Adherence factors includes fimbriae,pili, flagella, EPS(glycocalyx).(22)

Phase 2: Microbial and substrate adherence phase toform bridge by electrostatic attraction, covalent andhydrogen bonding, dipole interaction and hydrophobic

Invited review


55

interaction.(22,23,24)Phase 3: Specific microbial–substrate adherence phase

which involves bonding of adhesin or ligand on the bacterialcell surface.

There are numerous factors affecting bacterialattachment which includes pH, temperature variations, flowrate of fluid, nutrients, surface energy of the substrate,bacterial content, bacterial growth stage, bacterial cellsurface charge, and surface hyrophobicity.(25)

Step 3: Bacterial grow by co-adhesion of other organismsand biofilm expansion. Monolayer of microorganismsattracts secondary colonizers to form microcolonies by co-adhesion and coaggregation. These microcolonies aresimilar to towers with lateral and vertical growth ofmicroorganisms.(26)

Step 4: Detachment of sessile microorganisms i.e. presentwithin biofilm into their surroundings by seeding disposaland clumping dispersal.(14)

Seeding dispersal is a programmed detachment of

Difference between microorganisms in biofilm and planktonic counterparts

Sessile Bacteria Planktonic bacteria

1. Highly organized Not organized

2. Bound together by matrix Single

3. Attached to a solid surface with the help of fimbriae andfibrils Not attached to any surface

3. Colonies are surrounded by water channels Free floating

4. 1000 fold more resistant to phagocytosis, antibodies,antimicrobials. Lesser resistant than their counterparts

5. Compartmentalized depending on their metabolic andphysiological status within the biofilm Not organized

6. More virulent in terms of propagation of the infectiousprocess and able to survive without dividing. Less Virulent compared to their counterparts

7. Microcolonies are coated with impenetrablepolysaccharides Not coated

8. pH, ion concentration, nutrient availability and oxygen No such physical conditionssupply of the biofilm support the bacterial growth for planktonic bacteria

9. Gene transfer between the bacteria with the help of No such gene transfer takes place betweenfilaments, will convert them into highly virulent. planktonic bacteria

10. Exchange of nutrients, cross feeding, Signalingmolecules, protection and removal of harmful metabolic No such physicochemicalproducts take place between bacteria environment outside the biofilm

11. Some pioneer bacteria(Actinomyces) form scaffold forthe survival of extraradicular bacteria and maintainperiapical inflammatory process No such process takes place

12. Redox gradient formed within the biofilm for thesurvival of anaerobes when all available oxygen isconsumed by aerobic bacteria. No such changes take place

Planktonic bacterial cells caused by local hydrolysis of theextracellular polysaccharide matrix, and conversion of asubpopulation of cells into motile planktonic cells, whichcause persistent infection.

Clumping Dispersal: A physical detachment pathway inwhich a fragment of a microcolony, simply detaches fromthe biofilm and is carried by the bulk until it lodges in a newlocation and initiates a new sessile population. Detachmentcan be of two types Erosion (continous detachment of singlecell) and Sloughing (rapid detachment of biofilm).

Activities inside the biofilmBiofilm has overall complex architecture with niches and

also it possess different array of proteolytic enzymes. Maturebiofilm with increased nutrient, pH, metabolic products,signalling molecules and gaseous gradients possessesantimicrobial resistance. Oral biofilm is dominated byanaerobic bacteria because of redox gradient formed withinthe internal compartmentalization of biofilm.(27) Bacteriaresiding in a biofilm can communicate, exchange genetic

Endodontic biofilms-their formation and eradication: a review


56

materials and also acquire new traits. Communications inbiofilm are of two types which includes intraspeciescommunication(28) and inter species communication.(29)

Quorum sensing is intraspecies communication whichis mediated by low molecular weight molecules, which canalter the metabolic activity of neighboring cells andcoordinate the functions of resident bacterial cells within abiofilm.(30) Quorum sensing can also regulate microbialproperty such as virulence factors and extracellular DNAincorporation. Auto inducer system 2(AI -2) helps ininterspecies communication. This signal is released by manygram positive and gram negative microorganisms. Closeproximity of microorganisms facilitates gene transferbetween them.(31)

Biofilms in endodontics:Primary source of biofilm formation within the root canal

is the microorganisms present in the oral cavity. Theanatomical complexities in the root canal system provideshelter to microorganisms. Infectious processes in root canalgain sufficient power to cause subsequent destruction of thepulpal tissues only after biofilm formation. Firstidentification of biofilm was earlier reported by Nair 1987under transmission electron microscopy. The necrotic pulptissue becomes a favorable environment for microbialproliferation due to the presence of organic residue ornutrients, which act as substrate or culture medium.Intracanal microbiota existed as both as sessile and in theform of biofilm made up of cocci, rods and filamentousbacteria, single or multilayered, single/severalmorphological, multispecies adhered to dentinal wall of theroot canal with a distinct bacteria-dentin wall relationship,according the maturation stage of the biofilm.(31) Gram-negative bacteria are more frequent than Gram-positivebacteria. Facultative or strict anaerobic microorganisms aremore frequent than aerobic microorganisms and thepresence of bacilli and filaments is equivalent to that ofcocci. Intracanal biofilm are microbial biofilm formed onthe root canal dentine of infected tooth. A study haspresented evidence of E. faecalis colonization and biofilmformation in root canals of human teeth. To develop newtreatments to eradicate E. faecalis from persistent root canalinfections, the mechanisms through which the bacteriummaintains these infections must be understood. (32)

Endodontic biofilm can be of various categories1. Intracanal biofilm2. Extra radicular biofilm3. Periapical biofilm4. Foreign body centered biofilmMajor bulk of the organisms existed as loose collections

of cocci, rods, filaments and spirochetes apart from thisbacterial condensations were seen as palisade structuresimilar to dental plaque seen on tooth surface.(33) Extraradicular biofilm are root surface biofilms formed on rootsurface adjacent to the root apex of endodontically infected

teeth.(34) The extracellular matrix material of bacterial originwas also found. Bacterial biofilm can be seen beyond theapex of the root as bacteria in biofilm survive unfavorableenvironmental and nutritional conditions. Structurelesssmooth biofilm with multispecies bacteria was found onroot tips of surgically extracted teeth of refractory endodonticcases by Tronstad et al when examined under SEM and alsofound varying degree of extracellular matrix.(35)

Multilayered bacteria were seen embedded in a heavyextracellular matrix under SEM by Lomcali et al. on the roottips of asymptomatic apical periodontitis.(36) Calculus likedeposit was noticed at the root apex of extracted teeth afterpost treatment periapical periodontitis.(37)

Periapical microbial biofilms are isolated biofilms inthe periapical region of endodontically infected teeth. Theycan be seen even in the absence of root canal infections.Bacteria present in such biofilm should have the capacity toovercome host defence mechanisms and result in periapicallesions.(38)

Foreign body–centered biofilm is seen when bacteriaadheres to an artificial biomaterial surface and forms biofilmstructures.(39) It is also known as biomaterial centeredinfection. It is a major complication associated withprosthesis and also in an implant supported prosthesis.Biomaterial centered infection reveals opportunisticinvasion by nosocomial organisms. Bacterial adherence toa biomaterial can be described in three phases-Transportsof bacteria to biomaterial surface; Initial, non-specificadhesion phase; Specific adhesion phase. Coagulase–negative Staphylococcus, S. aureus, Streptococci,Enterococci, P.aeruginosa and fungi are commonly isolatedfrom infected biomaterial surfaces. In endodontics,biomaterial centered biofilm can be intraradicular orextraradicular depending upon the position of obturatingmaterial. Takemura N et al suggested Gram positivefacultative anaerobes have the ability to colonize and formextracellular polymeric matrix surrounding gutta-percha,serum plays a crucial role in biofilm formation.(40) Studieshave suggested that extraradicular microbial biofilm andbiomaterial centered biofilm are related to refractoryperiapical disease.(38)

Eradication of biofilm:The success of infected root canal treatment is dependent

on inactivation of microorganisms present in biofilm andplanktonic ambiance. Root canal preparation withantimicrobial agents, like chemical irrigants and/orintracanal dressings, can contribute for endodonticmicrobiota reduction(41). The microbiota of persistentapical periodontitis lesions is composed by diverse types ofmicroorganisms with biofilm-forming capacity, including P.acnes, S. epidermidis and F. nucleatum. Enterococcus faecalisis frequently associated with post-treatment endodonticinfections and refractory infections. In failed root canalslesions, adherence of bacteria to a substrate is the earlieststage in biofilm formation, eliciting the factors that links

Kundabala Mala


57

adherence of this bacterium to dentin would help inunderstanding its association with treatment.(42) A studyconducted by Anil Kishen et al. highlighted on the chemicalsthat alter the physicochemical properties of dentin willinfluence the nature of adherence, adhesion force, andsubsequent biofilm formation of E. faecalis to dentin. Therewere significant increases in adherence and adhesion forceafter irrigation of dentin with EDTA, whereas sodiumhypochlorite (NaOCl) reduced it. With the use ofchlorhexidine (CHX), the force of adhesion increased, butthe adherence assay showed a reduction in the number ofadhering bacteria. The irrigation regimen of EDTA, NaOCl,and CHX resulted in the least number of adhering E. faecaliscells. Biofilm disruption and cell viability were influencedby the species, their co-association in dual species biofilms,the test agent, and the duration of exposure. The effectivenessof NaOCl as an endodontic irrigant was reinforced. Henceaccording to many researchers contemporary root canalirrigants are able to disrupt and eradicate single- and dual-species biofilms.(43-44) A series of antimicrobial strategiesthat include chemomechanical preparation, intracanalmedication, and fluid tight root canal obturation have beentried to reduce endodontic microbiota.

The antimicrobial efficacy of ozonated water, gaseousozone, sodium hypochlorite and chlorhexidine in E. faecalisbiofilm was studied by Estrela, et al. (2007). The irrigationsolutions tested for 20 min were not sufficient to inactivateE. faecalis. (42) NaOCl was the most effective agent, capableof eradicating the biofilms after 1 minute at a concentrationof 0.00625%. CHX eradicated biofilm after 5 minutes at 2%.EDTA and citric and phosphoric acid solutions were noteffective against the biofilms at any concentration or timetested. Use of herbal irrigants have been suggested byPrabhakar et al. for eradicate E.Faecalis biofilm on toothsubstrate.(46) Jose et al. suggested that regular exchangeand use of large amounts of irrigant should maintain theeffectiveness and compensating for the effects ofconcentration.(47) Several studies have been developed withdifferent bacterial biofilm models to test the antimicrobialeffectiveness of endodontic medicaments. Investigationshave shown either antimicrobial success or failure ofintracanal medicaments on bacterial biofilm, as well as thepresence of biofilm on the entire external root apex surfacesin teeth with pulp necrosis and radiographically visibleperiapical lesions. The susceptibility of oral bacterial biofilmto antimicrobial agents indicates the need to reproduce invivo conditions where bacteria grow as biofilm on toothsurfaces. (48,49)

Nair, et al. (2005) suggested the need of non-antibioticchemomechanical measures to treat teeth with infected andnecrotic root canals so as to disrupt the biofilm.(6) However,the results of the critical review done by Carlos Estrela et al.confirmed that root canal preparation, with carefuldisinfection and use of intracanal substances that providegood antimicrobial efficacy, tissue dissolution capacity, andacceptable biocompatibility, will definitely improve the

prognosis of the treatment of apical periodontitis (44)

Method to identify and develope the modelsystem to develop endodontic biofilm

Confocal Laser Scanning microscope and fluorescencein situ hybridization are valuable tools for obtaining highresolution images and 3-D reconstruction of a variety ofbiological samples including their biofilm.(51)

The Calagary Biofilm Device (CBD) offers a new technologyfor the rational selection of antibiotics, effective againstmicrobial biofilms and for the screening of new effectiveantibiotic compounds.(50) The combined use of cetrimideand CHX provided good results against E. faecalis biofilms,and the alternating application more effective than thecombined mode of application.(52) Annie Shrestha et al.found in their study that nanoparticulates such asNanometric Bioactive Glass 45S5 are very efficient in reducingbiofilm bacteria, disrupt biofilm structure, and retain theantibacterial property even after aging. CS-np and ZnO-nppresent a potential approach in biofilm disinfection.(53)

Prof. Carlos Estrela suggested a model system to studyantimicrobial strategies in endodontic biofilms such asEnterococcus faecalis biofilms. Organism suspensions arecolonized in human root canals. Five milliliters of BrainHeart Infusion (BHI) should be mixed with 5 ml. of thebacterial inoculums (E. faecalis) and inoculated withsufficient volume to fill the root canal during 60 days. Thisprocedure should be repeated for every 72 h, always using24-h pure culture prepared and adjusted to No. 1 MacFarlandturbidity standard. Biofilm formation can be analyzed bySEM. We can observe E. faecalis consistently adhering tocollagen structure, colonized dentin surface, progressedtowards the dentinal tubules and forming a biofilm. Thisbiofilm model is viable for studies on antimicrobialstrategies and allows for a satisfactory colonization time ofselected bacterial species with virulence and adherenceproperties.(54)

ConclusionClinical success of root canal therapy mainly depends

on the eradication of microorganisms from the root canalsystem. Understanding the mechanism of disease process,microorganisms, their colonization and biofilm formationwill help us to disinfect the root canals. This review explainson biofilm formation and eradicating it from root canalsystem. Further research may contribute to develop newertechniques to completely render the root canal system diseasefree.

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25. Handley P et al.Surface structures found on Streptococcus sanguis strains maybe related to their ability to co aggregate with otheroral generaInfect Immun1985;(47):217-27

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27. Richards MJ et al . Nosocomial infections in medical intensive care units in theUnited states. National Nosocomial Infections Surveillance System. Crit CareMed 1999;27(5):887-7.

28. Spratt D,Pratten J . Biofilms and the oral cavity. Rev Environ Sci Bio/Technology2003;2:109-20.

29. Aamdol Scheie A, Peterson FC. The biofilm concept: consequences for futureprophylaxis of oral disease. Crit Rev Oral Biol Med 2004; 15:4-12.

30. Cvitkovitch DG, Li Y-H, Ellen R. Quorum sensing and biofilm formation inStreptococcal infections. J Clin Invest 2003; 112:1626-1632.

31. Nair PNR . Light and electron microscopic studies on root canal flora and periapicallesions. J Endod 1987;13: 29-39.

32. John W. Distel, DMD, MS, John F. Hatton, DMD, and M. Jane Gillespie, PhD.Biofilm Formation in Medicated Root Canals. J Endod 2002 28;10:689-93

33. Listgarten M . Formation of dental plaque and other biofilms In: Newman HN,Wilson M eds. Dental Plaque Revisited. Oral Biofilms in Health and Disease.Cardiff: Bioline, 1999: 187-210.

34. Frank SA, Barbour AG. Within –host dynamics of antigenic variation. InfectGenet Evol 2006;6(2): 141-6

35. Tronstad L, Barnett F , Cervone F . Periapical bacterial plaque in teeth refractoryto endodontic treatment. Endod Dent Traumatol 1990 ; 6:73-77.

36. Lomacali G, Sen BH, Cankaya H . scanning electron microscopic observationsof apical root surfaces of teeth with apical periodontitis. Endod Dent Traumatol1996;12:70-76.

37. Ricicci D, Martorano M , Bate AL , Pascon EA . Calculus-like deposit on the apicalexternal root surface of teeth with post treatment apical periodontitis: report oftwo cases. Int Endod J 2005;38:262-71.

38. Moine P, Abraham E. Immunomodulation and sepsis: impact of the pathogen.Shock 2004;22(4):297-308.

39. Wilson M. Susceptibility of oral bacterial biofilm to antimicrobial agents. J MedMicrobiol 1996;44(2):79-87.

40. Takemura N ,Noiri Y, Ehara A et al . Single species biofilm forming ability of rootcanal isolates on gutta-percha points. Eur J Oral Sci 2004;112:523-9.

41. Estrela C, Estrela CRA, Decurcio DA, Hollanda ACB, Silva JA. Antimicrobialefficacy of ozonated water, gaseous ozone, sodium hypochlorite andchlorhexidine in infected human root canals. Int Endod J. 2007;40:85-93.

42. Fujii R, Saito Y, Tokura Y, Nakagawa K-I, Okuda K, Ishihara K. Characterizationof bacterial flora in persistent apical periodontitis lesions. Oral Microbiol Immunol2009: 24: 502–505.

43. Anil Kishen, Chee-Peng Sum, Shibi Mathew, and Chwee-Teck Lim. Influence ofIrrigation Regimens on the Adherence of Enterococcus faecalis to Root CanalDentin.J Endod 2008;34:850 – 85.

44. Abdullah M, Ng Y-L, Gulabivala K, Moles D, Spratt DA. Susceptibilties of twoEnterococcus faecalis phenotypes to root canal medications. J Endod.2005;31:30-6.

45. Marýá Teresa Arias-Moliz, Carmen Marýá Ferrer-Luque, Miguel Espigares-Garcýá, PhD, Pilar Baca. Enterococcus faecalis biofilms eradication by rootcanal irrigants. J Endod 2009;35:711–714.

46. J. Prabhakar, M. Senthil Kumar, MS Priya, K Mahalakshmi, PK Seghal, VGSukumaran. Evaluation of antimicrobial efficacy of herbal alternatives Trephalaand green tea polyphenols, MTAD, and 5% sodium hypochlorite againstE.Faecalis biofilm formed on tooth substrate: An in vitro study. J Endod2010;36(1):83-86

47. Jose F. Sequeira, Kenio C. Lima, Fernando A C Magalhaes, Helio P. Lopes,Milton D. Uzeda. J Endod 1999;25(5):332-5

48. Spratt DA, Pratten J, Wilson M, Gulabivala K. An in vitro evaluation of theantimicrobial efficacy of irrigants on biofilms of root canal isolates. Int Endod J.2001;34:300-7.

49. Carlos Estrela, Gilson Blitzkow Sydney, José Antonio Poli Figueiredo, CyntiaRodrigues de Araújo Estrela. antibacterial efficacy of intracanal medicamentson bacterial biofilm: a critical review. J Appl Oral Sci. 2009;17(1):1-7.

50. H. Ceri, m. E. Olson, c. Stremick, read, d. Morck, and a. Buret. The CalgaryBiofilm Device: New Technology for Rapid Determination of AntibioticSusceptibilities of Bacterial Biofilms. J. Clin. Microbiol. 1999;37( 6) :1771-6

51. Dige I, Nilsson H, Kilian M, Nyvad B. In situ identification of streptococci andother bacteria in initial dental biofilm by confocal laser scanning microscopyand fluorescence in situ hybridization. Eur J Oral Sci, 115(6): 459-67.

52. Marýá Teresa Arias-Moliz, Carmen Marýá Ferrer-Luque, Marýá PalomaGonza´lez- Rodrý´guez, Mariano Jose´ Valderrama, and Pilar Baca. Eradicationof Enterococcus faecalis Biofilms by Cetrimide and Chlorhexidine .J Endod2010;36:87–90.

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54. Carlos Estrela, Gilson Blitzkow Sydney, José Antonio Poli Figueiredo, CyntiaRodrigues de Araújo Estrela. A model system to study antimicrobial strategiesin endodontic biofilms. J Appl Oral Sci. 2009;17(2):87-91

Kundabala Mala


59

details consent obtained and then examined for dentalcaries.

Inclusion Criteria

Study group consisted of 75 Healthy Adult subjectsranging in age from 25-50 years having DMFT score of > 5.

Control group consisted of 25 Healthy caries-free adultsubjects ranging in age from 25-50 years.

Exclusion criteria

Patients with periodontitis, patients undergoingchemotherapy or radiotherapy, patients with systemicdisease of vital organs, diabetic patients, hypertensive’s andpatients with history of long term medication were excludedfrom the study.Sampling Method : Purposive sampling

Sample Size : 100 (75 study group + 25 control group)A detailed case history of the patients was taken. Patient’s

consent was obtained. Patient’s Body Mass Index wascalculated using the formula:

BMI = weight (kg)/height2 (m2)Patient’s saliva is collected to check the following using

a saliva check kit :

Correlation between vitamin c level and dental caries – A clinical study* Mithra N. Hegde, ** Nidarsh D. Hegde , *** Sucheta Shetty, **** Ann Moany, **** Vinod R. Jathanna

* Senior Professor and Head** Professor, A.B.Shetty Memorial Institute of Dental Sciences, Mangalore*** Professor in Biochemistry, K.S. Hegde Medical Academy, Deralakatte, Mangalore,

A.B.Shetty Memorial Institute of Dental Sciences, Deralakatte, Mangalore**** Former P.G. Student, A.B.Shetty Memorial Institute of Dental Sciences,

Deralakatte, Mangalore

Introduction: In vitro ascorbic acid has either a bactericidal or a bacteriostatic effect. Therefore there is a needto know whether a change in ascorbic acid, appearing as a change in plasma level of ascorbic acid, is reflectedas altered cariogenic flora or as changes in its components and as altered caries prevalence.

The aim of the current study was to evaluate the correlation between ascorbic acid and dental caries andpredisposing factors such as salivary levels of mutans streptococci, Body Mass Index and the nature andquality of saliva- flow, volume, viscosity, pH, and buffering capacity.

Methods: 100 healthy adult subjects were selected for the study. 5ml of the patient’s blood was collected. 1mlof the blood was used for blood grouping and estimation of total count, differential count, and hemoglobinlevel. 4 ml of the blood was utilized for estimation of vitamin C level using Dinitrophenyl hydrazine method(DNPH).

For the estimation of streptococcus mutans level, swab was collected from the carious lesion and inoculatedin SL Agar for 24 hours. Colonies were identified by gram staining method.

Results: The serum vitamin C level was significantly higher in the control group than in the study group. Therewas no significant between the number of Mutans streptococci and serum vitamin C level. No significantcorrelation was found between DMFT and Body Mass Index. Salivary parameters as tested by the saliva checkkit showed no correlations with caries prevalence scored using DMFT index.

Conclusion: Serum vitamin C level decreases with increase in caries activity. Vitamin C acts by neutralizing thefree radicals.

Abstract

Introduction

High prevalence and incidence of caries are associatedwith high levels of Streptococci mutans and Lactobacilli inthe mouth. Therefore, the levels of these bacteria are used intesting of caries prediction(1-7).

The rate of salivary secretion is considered to be animportant factor in protection against dental caries, as thebuffer capacity of saliva increases along with increase insecretion rate and because saliva rinses off acids ofnutritional origin or arising from bacterial action(8,9).

In vitro ascorbic acid has either a bactericidal or abacteriostatic effect. Therefore there is a need to knowwhether a change in ascorbic acid, appearing as a change inplasma level of ascorbic acid, is reflected as alteredcariogenic flora or as changes in its components and asaltered caries prevalence(10-13).

The aim of the current study was to evaluate thecorrelation between ascorbic acid and dental caries andpredisposing factors such as salivary levels of Streptococcimutans, Body Mass Index and the nature and quality ofsaliva-flow, volume, viscosity, pH, and buffering capacity.

Materials and methods

Participant recruitmentPatients visiting the OPD in the Department of

Conservative Dentistry and Endodontics, A.B.Shetty MemorialInstitute of Dental Sciences were informed about the study

Original Scientific Articles


60

Mithra N. Hegde

Resting saliva – hydrationResting saliva – viscosityResting saliva – pHStimulated saliva – quantityStimulated saliva – buffering capacity

Following tests were done in research laboratory :Blood grouping,Hemoglobin level, Total count, differential

count, vitamin C level, Streptococcus mutans level.In the lab 5 ml of the patient’s blood is collected. 1 ml of

the blood is used for blood grouping and estimation of totalcount, differential count, and hemoglobin level. 4ml of thecollected blood is kept for coagulation for half an hour.Serum is separated by centrifugal method for 10 minutes at3000 rpm. The serum is utilized for estimation of vitamin Clevel using Dinitrophenyl hydrazine (DNPH) method.

Reagents used : 2, 4 dinitrophenylhydrazine, 6% TCA,Acid washed norit, 85% sulfuric acid, thiourea.

For the estimation of Streptococcus mutans level, swabis collected from the carious lesion using a wooden swabstick and inoculated in SL Agar for 24 hours. Colonies areidentified by gram staining method.

ResultsThe serum vitamin C level was significantly higher in the

control group than in the study group (table 1).There was no significant correlation between the number

of Streptococci mutans and serum vitamin C level (table 2).No significant correlation was found between DMFT and

Body Mass Index (table 2) Salivary parameters as tested bythe saliva check kit showed no correlations with cariesprevalence scored using DMFT index (table 3).

Discussion

The relationship between the plasma level of vitamin cand number of carious lesions in the mouth is poorlyunderstood4. Large numbers of free radicals are produced

during the process of dental decay. The numbers of freeradicals vary directly with the degree of activity of thecaries(14-19).

A free radical is any atom with at least one unpairedelectron in the outer most shell, and is capable of independentexistence. Free radicals are very unstable and react quicklywith other compounds, trying to capture the needed electronto gain stability. Normally, the body can handle free radicals,but if antioxidants are unavailable, or if the free radicalproduction becomes excessive, damage can occur(5).

Vitamin C is the most abundant water soluble antioxidantin the body. Antioxidants neutralize free radicals by donatingone of their own electrons. Dinitrophenyl hydrazine method(DNPH) measures the total L-Ascorbic acid, dehydroascorbicacid and 2,3-di ketogluconic acid, and is based upon themeasurement of the color of the product formed by thecoupling of 2,4-dinitrophenylhydrazine with the ketonicgroup of oxidized ascorbic acid(18,19).

Salivary levels of Streptococci mutans do not alwaysindicate their presence in deeper layers of plaque, which islocated at the most caries prone surface areas (20, 21, 22,9).Ascorbic acid content in plasma seems to have no effect onthe levels of these bacteria. Available evidence suggests thatintake of ascorbic acid has little or no effect on oral healththrough saliva (11).

A small amount of salivary flow can influences cariesformation by decreasing rinsing of the dentition, but salivaryflow did not differ between groups. Salivary parameters astested by the saliva check kit showed no correlations with

Table I Serum Vitamin C level in the control group and the study group.

Group N Mean Std. Deviation Std. Error Mean

Concentration ofVitamin C Control 25 8.5480 1.01822 .20364

Concentration ofVitamin C Case 75 3.8320 .92242 .10651

Table 2. Correlation between serum vitamin C level andstreptococcus mutans level, and Body Mass Index and DMFT

Variables Correlation p-valuesco-efficient

Vitamin C andStreptococcus mutans 0.073 0.535

DMFT and BMI 0.126 -0.280

Table 3. Correlation of salivary parameters with caries prevalence

Variables Correlation co-efficient p-values

DMFT and restingsaliva-hydration -0.159 0.172

DMFT and restingsaliva-viscosity -0.096 0.415

DMFT and restingsaliva -pH -0.098 0.398

DMFT and stimulatedsaliva -quantity - -

DMFT and stimulatedsaliva -buffering capacity -0.071 0.375


61

caries prevalence scored using DMFT index. This findingwas possibly due to the lack of sensitivity of the DMFT index.

ConclusionSerum vitamin C level decreases with increase in caries

activity. Vitamin C acts by neutralizing the free radicals.

References1. Stecksen-Blicks C. Salivary counts of lactobacilli and streptococcus mutans in

caries prediction. Scand J. Dent. Res. 1985;93:204-12.2. Bolton RW, Hlava GL. Evaluation of salivary IgA antibodies to cariogenic

microorganisms in children. Correlation with dental caries activity. J DentRes.1992;61(11):1225-8.

3. Camling E, Ghanberg L, Krasse B. The relationship between IgA antibodies tostreptococcus mutans antigens in human saliva and breast milk and the numbersof indigenous oral streptococcus mutans. Arch Oral Biol 1987; 32(1):21-5.

4. Emilson CG, Krasse B. Support for and implications of the specific plaquehypothesis. Scand J. Dent Res 1985;93(2):96-104.

5. Fontana M, Buller TL, Dunipace AJ, Stookey GK, Gregory RL. An in vitromicrobial-caries model used to study the efficacy of antibodies to streptococcusmutans surface proteins in preventing dental caries. Clin Diagn Lab Immunol2000;7(1):49-54.

6. Krasse B. Interpretation and use of microbiologic findings in dental caries. OralMicrobiol Immunol 1986;1:85-6.

7. Tenovuo J, Jentsch H, Soukka T, Karhuvaara L. Antimicrobial factors of salivain relation to dental caries and salivary levels of mutans streptococci. J BiolBuccale 1992;20(2):85-90.

8. Tukia-Kulmala H, Tenovuo J. Intra and inter-individual variation in salivary flowrate, buffer effect, lactobacilli, and mutans streptococci among 11- to 12-year-old school children. Acta Odontol Scand 1993; 51(1):31-7.

9. Battino M, Ferreiro Ms, Gallardo I. The antioxidant capacity of saliva. Journalof clinical periodontology. 2002;29:189-194.

10. Bates H F., Hughes R E, Hurley R J. Ascorbic acid status in man. Measurementsof salivary, plasma and white blood cell concentration. Arch Oral Biol 1972;17:1017-20.

11. Leggott P J, Robertson P B, Rothman D L, Murray P A, Jacob R A. Response oflingual ascorbic acid test and salivary ascorbate levels to change in ascorbicacid intake. J Dent Res. 1986; 65:131-4.

12. Woolfe S N, Hume W R, Kenney E B. Ascorbic acid and periodontal disease : Areview of the literature. J West Soc.Periodontol 1980;28:44-56.

13. Parviainen M, Nyyssonen K, Penttile I. A method for routine assay of plasmaascorbic acid using high performance liquid chromatograph. J. Liq. Chromatogr1986;9:2085-97.

14. Battino M, Ferreiro Ms, Gallardo I. The antioxidant capacity of saliva. Journalof clinical periodontology. 2002;29:189-194.

15. Uberos J. Alarcon J.A., Penalver M.A. Influence of the antioxidant content ofsaliva on dental caries in an at risk community. British dental journal2008;205:E5.

16. Vincent F, Lisanti, Eichel B. Antioxidant inhibition of experimentally inducedcaries in hamsters. Journal of dental research. 1963;42(4):1030-1035.

17. Jordan H.V., Bowler A.E., Berger N.D. Testing of antioxidants againstexperimental caries in rats. Journal of dental research. 1961;40(5)878-883.

18. Koracevic D, Koracevic G, Djordjevic V. Method for the measurement ofantioxidant activity in human fluids. Journal of clinical pathology 2001;54,356-361.

19. Huang D, Ou B, Prior R.L. The chemistry behind antioxidant capacity assays.Journal of Agricultural food chemistry 2005;53:1841-1856.

20. Hajishengallis G, Koga T, Russell M W. Affinity and specificity of the interactionsbetween streptococcus mutans antigen I/II and salivary components. J. Dent.Res. 1994;73:1493-1502.

21. Lamont R.J., Demuth D R, Davis C.A. Malamud D, Rosan B. Salivary-agglutinin-mediated adherence of streptococcus mutans to early plaque bacteria. InfectImmun. 1991;59:3446-3450.

22. Loesche W J. Role of Streptococcus mutans in human dental decay. MicrobialRev. 1986: 50:353-380.

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Correlation between vitamin c level and dental caries – A clinical study


62

The aim of the present study was to evaluate usingscanning electron microscopy, the quality of resin tags formedat the resin-dentin interface by self-etching adhesives andto evaluate the effect of additional acid etching on resin tagformation of self-etching adhesives. This in-vitro study isdesigned to test the hypothesis that the complete removal ofsmear layer prior to the use of self-etching adhesives wouldresult in better resin tag quality.

Methodology:The present study was conducted at the Department of

Conservative Dentistry and Endodontics, A. B. ShettyMemorial Institute of Dental Sciences, Mangalore; andDepartment of Metallurgy, Indian Institute of Sciences,Bangalore.

Forty-six, freshly extracted caries free human molars werecollected, stored, disinfected and handled as per therecommendations and guidelines laid down by Occupational

Effect of additional acid etching on resin tag formation with self-etchingadhesives - A scanning electron microscopy study

* Sunil M. Eraly, ** Mithra N. Hegde, *** Priyadarshini Hegde

* Ph.D. Student, NITTE university, Mangalore,Reader, Conservative Dentistry and Endodontics, Malabar Dental College andResearch Centre, Mudur PO, Edappal, Malapuram - 679578

** Senior Professor and Head,*** Professor, Department of Conservative Dentistry and Endodontics, A.B. Shetty

Memorial Institute of Dental Sciences, Deralakatte, Mangalore, Karnataka.

Aims and Objectives: To observe the quality of resin tags formed at the resin-dentin interface produced by afifth generation adhesive as compared to two self-etching adhesives and to evaluate the effect of additionalacid etching on the resin tag formation of self-etching adhesives using Scanning Electron Microscopy.

Materials and Methods: Standardized Class I cavities were prepared in all forty six freshly extracted humanmaxillary and mandibular molars and randomly divided into: Group I: 35% Phosphoric acid etch followed byExcite DSC; Group IIA: Clearfil Liner Bond 2V; Group II B: 35% Phosphoric acid etch and Clearfil Liner Bond 2V;GroupIIIa: Adper Prompt; Group IIIB: 35% Phosphoric acid etchand Adper Prompt. All specimens were restoredwith a packable composite (Filtek P60), sectioned, gold sputtered and examined in a Scanning ElectronMicroscope. The results were tabulated and analyzed using Analysis of Variance (ANOVA) and Tukey HonestlySignificance Difference test.

Results: The values obtained are listed in decreasing order:

Resin Tag Penetration- Group IIIB: 40.18µm; Group IIIA: 28.23µm; Group I: 25.13µm; Group IIB: 15.41µm;Group IIA: 5.7µm.

Hybrid Layer thickness- Group IIIB: 3.27µm; Group IIB: 2.83µm; Group I: 2.7µm; Group IIIA: 2.31µm;Group IIA: 0.64µm.

Statistical analysis of the data using ANOVA showed very highly significant values.

Conclusions: Dentin pretreatment with 35% phosphoric acid prior to application of self-etch adhesives re-sulted in thicker and longer resin tags and thicker hybrid layer.

Keywords: Dentin bonding agents; Total etch; Self-etch adhesive; Hybrid layer; Scanning Electron Microscope.

Abstract

Introduction

Adhesive dentistry has revolutionized restorative dentalpractice during the last four decades. The introduction ofnewer bonding systems, the sixth generation self-etchingprimers, being single step bonding agents, has reduced thechair side time to do a composite restoration. These bondingsystems may be classified as mild, moderate or aggressivebased on their ability to penetrate the dentin smear layerand their depth of demineralization into subsurface dentin.The more aggressive system completely solubilised the smearlayer and smear plugs and formed hybrid layers withthickness approaching those of phosphoric acid conditioneddentin(1).

Scanning Electron Microscopy (2) is a commonly usedmethod along with other various methods of observationlike Transmission Electron Microscopy and Confocal OpticalMicroscopy. The combination of high resolution and extensivemagnification range and high depth of field makes scanningelectron microscopy uniquely suited for analyzing surfaces.It is an indispensable tool in material science research anddevelopment. Scanning electron microscopy has become themost popular and easiest tool to morphologically examinethe bonding mechanism.


63

Safety and Health Administration (OSHA) and Centres forDisease Control and Prevention (CDC). All the teeth wereexamined and teeth with surface defects and cracks wereeliminated. The teeth were pumice washed and then randomlydistributed among groups. Protocols in cross infectioncontrol as per OSHA regulations in storing, surfacing andutilization were observed.

Standardized Class I cavity preparations were done inall the teeth using a No. 245 carbide bur with a high speedairotor hand piece under air/water spray. The size of thepreparation was made proportional to the dimensions ofthe tooth to minimize variations resulting from tooth size.The specimens were randomly divided into a control groupand two experimental groups.

Control Group:Group I (Six Specimens):

All the prepared specimens in this group were etchedwith 35% phosphoric acid for 15 seconds and thereafter,rinsed for 15 second and blot dried to remove excess water.Two coats of the single component total etch adhesive, ExciteDSC was applied to the entire preparation and light curedaccording to manufacturer’s instructions.

Experimental Groups: All the prepared specimens in thisgroup were cleaned and air dried for 5 seconds to removeexcess water.

Group II (Twenty Specimens):

Sub Group II A (Ten Specimens): Clearfil Liner Bond 2Vwas applied to the entire preparation and light curedaccording to manufacturer’s instructions.

Sub Group II B (Ten Specimens): Acid etching was doneusing 35% phosphoric acid for 15 seconds and thereafterrinsed and air dried for 5 seconds to remove excess water.Clearfil Liner Bond 2V was applied to the entire preparationand light cured according to manufacturer’s instructions.

Group III (Twenty specimens)

Sub Group III A (Ten Specimens): Adper Prompt wasapplied to the entire preparation and light cured accordingto manufacturer’s instructions.

Sub Group III B (Ten Specimens): Acid etching was doneusing 35% phosphoric acid for 15 seconds and thereafterrinsed and air dried for 5 seconds to remove excess water.Adper Prompt was applied to the entire preparation andlight cured according to manufacturer’s instructions.

All the class I preparations were then restored withpackable composite in an oblique incremental layeringtechnique of 1 mm each, curing each layer for 40 secondsand stored under room temperature. They were sectionedusing diamond impregnated disc, under running water,demineralized with 35% Phosphoric acid for 30sec, and thenrinsed and deproteinized using 3% sodium hypochlorite for10min, then rinsed and dried. The specimens were mountedon brass stubs gold sputtered in a gold sputtering machineand examined in a Scanning Electron Microscope (SEM). Theresults obtained were tabulated and statistically analyzed.

Results:

The data of the present study showed that mean valuesfor Group III B, Adper Prompt - self-etch adhesive withadditional acid etching, displayed the highest depth ofpenetration of resin tags.

Group II A, Clearfil Liner Bond 2V - self-etch adhesive,showed the least depth of penetration of resin tags.

Group I, bonded with Excite DSC a fifth generation dentinbonding agent showed mean length of resin tags close toGroup III A, Adper Prompt - self-etch adhesive when usedalone.

Statistical Analysis: The data of the present studysubjected to statistical analysis showed that:

Table I: Inter-group comparison of mean length of resintag penetration using Analysis of Variance (ANOVA) showedthe values to be very highly significant.

Table II: Inter-group comparison of mean length of resintag penetration using Tukey Honestly Significance DifferenceTest showed very highly significant results in all inter-groupcomparisons except when Group I was compared to GroupIII a which showed statistically significant results.

Discussion:Today we are in an age of ‘Adhesive Dentistry’. Buonocore’s

discovery of the acid-etch technique led to these changes inthe present day practice of dentistry. Traditional mechanicalmethods of retaining restorative materials have beenreplaced to a large extent by tooth conserving adhesiverestorative techniques.

Effect of additional acid etching on resin tag formation with self-etching

adhesives - A scanning electron microscopy study


64

Sunil M. Eraly

Dentin is the fundamental substrate of restorativedentistry and its properties and characteristics are keydeterminants of nearly all restorative, preventive and diseaseprocesses of teeth. Improved understanding of the nature ofthe dentin has important consequences for today’s dentalprocedure and should lead to new methods to preserve andprotect teeth and repair defects brought on by disease ortrauma. Current research is focused on newer methods toproduce a bond between restorative materials and dentin.Enamel has a long track record of being an excellent bondingsubstrate with composite resins(3). Number of studies hasbeen carried out on enamel, proving it to be an excellentbonding substrate. However dentin bonding has been quiteperplexing and little research has been conducted againstthe substrate.

Various bond strength studies have shown that self-etching adhesives are inferior to fourth and fifth generationbonding systems. D.H.Pashley(4) demonstrated etchingpatterns and bonds to enamel with these systems have beenweaker compared to previous generations. E.A.Glasspooleet al(5) proved using Scanning Electron Microscopy and bondstrength analysis that the bond to enamel of self-etchingadhesives can be improved by pretreating enamel with 37%phosphoric acid. He further stated that the depth ofresinpenetration was also ditrectly related to the bondstrength. K.T. Jang et al (6) evaluated the effect of additionalacid etching on resin tag formation of three self-etchingprimers and a one bottle adhesive. They reported thatadditional etching of dentin improved thickness and depthof penetration of resin tags.

In the present investigation we have made an attempt tocompare ultramorphologically, one fifth generation bondingsystem and two self-etching adhesive systems and the effectof additional acid etching on resin tag formation of self-etching adhesives. The Scanning Electron Microscopicpictures that are produced were focused on the length andthickness of resin tags.

All the scanning electron microscopic pictures that areseen in published papers or product profiles that show long

resin tags try to imply that this is desirable and contributeto bond strength. Although deep dentin exhibited more tagsthan superficial dentin, the bond strength of resin compositeto deep dentin was generally lower than those made tosuperficial dentin(7,8,9). According to D.H.Pashley et al (10)resin tags only contribute to the bond strength if the resinactually bond to the wall of dentinal tubules. As most clinicalcavity preparation include both superficial and deep dentinit would be desirable to have dentin bonding agents thatbonded equally well to both types of dentin.

In this present invitro investigation we could record fairlydeep resin tags penetration in two groups which includedExcite DSC (25.13µm) as a representative of fifth (total-etch)generation of bonding system and the self-etching adhesiveAdper Prompt (28.23µm). Clearfil Liner Bond 2V (5.7µm)showed lesser resin tag penetration. The values obtainedare in correlation with D.H. Pashley(1) who showed the moreaggressive self etch adhesive has better depth of penetration.However after additional acid etching in the two self-etchadhesive groups we observed deeper resin tags with ClearfilLiner Bond 2V averaging 15.41µm and Adper Promptaveraging 40.18µm. Ry Andia-Merlin11 in their study havedemonstrated resin tags upto 100 µm in length. Clearfil LinerBond 2V demonstrated comparatively lower mean values asfar as the depth of penetration of the resin tags wasconcerned. With Adper Prompt we could record maximumlength of resin penetration while with Excite DSC we observedcomparatively lower values. The additional observationmade in the study was, that additional acid etching alongwith sixth generation self-etch adhesives resulted in thickerand longer resin tags which may contribute to additionalbond strength.. The increase in the thickness of resin tagsand microtags observed in the SEM pictures may be a factorwhich indicates bonding to peritubular dentin within thedentinal tubules.

Ultramorphological findings with recent generationadhesives indicated evidence to fact that resin tags docontribute to the final bond strength due to the supplementaryretention and sealing offered by the formation of microtagsand lateral branching from the main tubules. Such microtagsin lateral tubule braches show hybridization as well as aphenomenon defined as lateral tubule hybridization(13).

In our study, we observed lateral tubule hybridizationwith Excite DSC and Adper Prompt. After Additional etching,all specimens in self-etching adhesive groups showedmicrotags. Franklin R. Tay (12) studying effect of smear layeron bonding self etching primer to dentin concluded that selfetching primers create thin hybrid layer that incorporate thesmear layer. Their study demonstrated formation of thinhybrid layer in comparison to the smear layer thickness andboth hybrid layer and smear layer may function as a singleunit during loading without separation.

Concerning the result that are obtained we wish toemphasize that we have considered only two parametersthat is length and thickness of resin tags and thickness of


65

hybrid layer of two self-etching adhesives and fifthgeneration adhesive. We do not want to draw conclusion onthe efficacy of a particular fifth generation dentin adhesiveor self-etching adhesives by just looking into these twoparameters. Before rank ordering a particular dentinadhesive system, there are several methods of evaluation.Scanning Electron Microscopy is one of known methods tocompare and correlate bonds strengths studies. In all in-vitro studies there are several parameters involved includingthe collection of samples, freshness of the specimen, andmethod of storing, surfacing and utilization of thesespecimens.

In addition in-vivo conditions are different and lab in-vitro investigations are mostly dependant upon by themanufactures for highlighting the efficacy of their products.The self etching adhesives are fairly new in the commercialmarket, a system that is simple, efficacious with simple steps

& less technique sensitive. However, commercially availabledentin bonding system including the sixth generation self-etching adhesives have demonstrated number of weaknessesin both in-vitro & in-vivo studies. The quest for an idealbonding system is on globally, to arrive at the ultimatebonding system which would seal the exposed cellularcomponents within dentinal tubules & also result inbiological compatible material.

More research is required to improve the bonding efficacyof self etching adhesives to different clinical substrates andto evaluate their long term success.

Conclusion:In the present study a Scanning Electron Microscopic

evaluation was carried out to demonstrate the depth andthickness of the resin tag penetration in dentin of the fifthgeneration bonding system and two sixth generation dentin

SEM of Group I SEM of Group II a

SEM of Group II b SEM of Group III a

Effect of additional acid etching on resin tag formation with self-etching

adhesives - A scanning electron microscopy study


66

bonding system and effect of additional acid etching on resintag formation of self-etch adhesives, and the followingconclusions were drawn.

Additional acid etching of dentin prior to application ofself-etch adhesives resulted in improved quality of resintags compared to self etching adhesives when used alone.

References:1. Franklin R. Tay, David H. Pashley. Aggressiveness of contemporary self-etching

systems. I: Depth of penetration beyond dentin smear layers. Dental Materials2001; 17: 296-308.

2. Boyde A, Knight PJ. The use of Scanning Electron Microscopy in clinical dentalresearch: British Dental Journal 1969,October 7: 313-321.

3. Bowen RL, Marjenhoff W.A. Development of an adhesive bonding systems.Operative Dentistry 1992; suppl 5: 75-80

4. David H. Pashley, Franklin R. Tay. Aggressiveness of contemporary self etchingadhesives part II: etching effects on unground enamel. Journal of DentalMaterials 2001; 17: 430 – 444.

5. Eileen A. Glasspoole, Robert L. Erickson, Carel L. Davidson. Effect of enamelPretreatments on bond strength of compomer. Dental Materials 2001; 17:

Sunil Earaly

402-408.6. Jang, KT, Gracia-Godoy F. Effect of additional acid etching on resin tag formation

of self-etching adhesives. Journal of Dental Research 2002, Special Issue A;(21): A-247. Abstract number: 1894.

7. Suzuki T, Finger WJ. Dentin adhesives: site of dentin verses bonding ofcomposite resins. Dental Materials 1988; 3: 379-383.

8. Nakamichi I, Iwaku M, Fusayama T. Bovine teeth as possible substitutes in theadhesion test. Journal of dental research 1983; 62: 1076-1081.

9. Causton BE. Improved bonding of composite restorative to dentin. British dentaljournal 1984; 156: 93-95.

10. D.H. Pashley, B. Ciucchi, H. Sano. Dentin as a bonding substrates. Vortrag aufder Jahrestagung der D.G.Z. 1994.

11. Ry Andia-Merlin, Garone-Netto N, Ve Arana-Chavez. SEM evaluation of theinteraction between a three- step adhesive and dentin. Operative Dentistry2001; 26: 440 – 444.

12. Franklin R. Tay, Hidehiko Sano, Ricardo Carvalho, Edna L. Pashley, David H.Pashley. An ultra structural study of the influence of acidity of self –etchingprimers and smear layer thickness on bonding to intact dentin. Journal ofAdhesive Dentistry 2000; 2:83-98.

13. Bart van Meerbeek, Marcos Vargas. Microscopy investigations techniques,results, limitations. American Journal of Dentistry 2000; 13: 3D –18D.

SEM of Group III b Latral hibridization


67

effect of monomers that are present in dentin bondingsystems(5).

Histopathological observations suggested that dentin-bridge formation may occur following three patterns: (a)within 14 days from the periphery of the residual dentinchip at the wound surface; (b) within 14 days from theperiphery of the cavity floor and with formation of reparativedentin by stimulation during the cavity preparation; (c)within 30 days from the wound surface(6,7). Pulp reactioncan originate from multiple factors such as the preparationtechniques, the toxicity of the material, and bacterialcontamination at the material-cavity wall interface(7).

The purpose of this work is to evaluate and compare theeffect of two bonding agents on the pulp and to measure theodontoblastic activity near these materials to assess theirbiocompatibility.

Aims & objectives1) To evaluate the immediate cellular events that occur

in direct pulp exposure which is capped by a fourthgeneration bonding agent (Stedbond), a fifth generationbonding agent (Single Bond) and a hard setting calciumhydroxide (Dycal).

2. To evaluate and compare percentage of

Comparative evaluation of odontoblastic activity followingdirect pulp capping with two bonding agents and

calcium hydroxide in humans - A histological analysis* Sreeja Sreedhar, ** Ramesh Kumar M.

* Reader, Dept. of Conservative Dentistry and Endodontics, Sri Sankara DentalCollege, Varkala, Trivandrum.

** Professor and HOD, Dept. of Conservative Dentistry and Endodontics,Govt. Dental College, Kozhikode.

Aim: Pulp capping is the simplest form of vital pulp therapy. It is found that prognosis of direct pulp cappingdepends on the restorative procedure, bacterial microleakage and biocompatibility of the materials used.

Recently bonding agents also, because of their adhesiveness is suggested as pulp capping material. Howeverall bonding agents may not produce the same result due to their varying composition and presence of certaincytotoxic materials in them.

This study compares odontoblastic activity and other immediate cellular events near the machanical pulpexposure capped with three different materials and thus determine their biocompatibility.

Materials and Methods: A fifth generation bonding agent ‘Single Bond’ and a fourth generation bonding agent‘Stedbond’ were used as the test group and Dycal - a hard setting calcium hydroxide as the control group. Thestudy was conducted in 30 healthy permanent premolars in 12 patients and were extracted after 15 days formicroscopic examination.

Result: All the materials Showed odontoblastic activity near the pulp capping. But it was significantly lower inSingle Bond compared to other two materials. Sted Bond showed comparable result with that of Dycal.

Conclusion: Single Bond cannot be considered as a biocompatible material for direct pulp capping. Sted Bondappears promising as direct pulp capping material, but requires further long term study.

Abstract

IntroductionDirect pulp capping is the dressing of an exposed pulp

with the aim of maintaining pulpal vitality(1). The materialsused for direct pulp capping should be biocompatible(2).

It has been realized that the variable prognosis of vitalpulp capping is a restorative issue. The major cause of post-operative inflammation and pulp necrosis following directpulp capping are thought to be non-sterile procedures andbacterial microleakage(3).

Calcium hydroxide has been accepted as the best materialdue to its proven capacity to obtain high percentages ofsuccess. But due to the lack of adherence to tooth structureand the presence of multiple tunnel defects in the dentinbridges formed beneath them, search for better pulp cappingmaterial continued(3).

Proponents of adhesive bonding as pulp capping agentspoint out that germ-free study have shown rapid healing ofpulp even when bonding agents are placed directly on pulpaltissue(4). However, the choice of the adhesive resin systemis important for the success of direct pulp-capping .Importantly, the adhesiveness and differences in materialcontent may affect the results(3).

There are also reports of post-placement tooth sensitivity,local immunological effects, apoptotic reactions, long-termpulpal inflammation, systemic estrogenic effects andpossible carcinogenic effect.

Research is available regarding the in vitro cytotoxic


68

Sreeja Sreedhar

odontoblastic activity near mechanical pulp exposurecapped with these three materials and thereby assessingtheir biocompatibility.

Materials & methodsIn the present in vivo study, 30 non-carious healthy

permanent premolars scheduled for extraction fororthodontic reasons from 12 healthy patients were selectedfrom Department of Orthodontics, Government DentalCollege, Calicut. The selected patients were in the age groupof 13-16 years. The study protocol was approved by theethical committee of Medical College, Kozhikode

Following materials were used as direct pulp cappingagents in patients selected as per the criteria given below.

1. Single Bond (3M ESPE): A fifth generation dentinbonding agent, containing BisGMA, HEMA, water, ethanoland polyalkenoic acid copolymer.

2. Stedbond (Stedpharma): A fourth generation dentinbonding agent. It is a two bottle system with acetone basedprimer containing HEMA and BisGMA as adhesive.

3. Dycal: A hard setting calcium hydroxideThe samples were divided into 3 groups of 10 specimens

each:Group I (experimental group) - Direct pulp capping with

stedbond and restored with Curex Microhybride Light CureComposite (DPI).

Group II (experimental group) - Direct pulp capping withsingle bond and restored with Curex Microhybride Light CureComposite (DPI).

Group III (Control group) - Direct pulp capping with Dycal and restored with Curex Microhybride Light Cure Composite(DPI) after applying bonding agent - Gluma Comfort Bond(Heraeus Kulzer).

The clinical procedure was as follows

After obtaining adequate anesthesia with local anesthetic(lignocaine HCl 2% wt / vol, with adrenalin base of 0.001%wt / vol) rubber dam is applied to isolate the teeth.Conventional class I cavities are prepared on the teeth withstraight bur with high speed under air water coolant. A pulpexposure of 0.5 to 1.0 mm diameter is carefully made with ¼size round bur at slow speed with air water spray to limit the

pulp injury.The exposed pulp surface is rinsed with saline solution

and dried, then treated with freshly prepared 0.5% NaOCland 3% H

2O

2 for disinfection and hemostasis.

After obtaining adequate hemostasis, the cavity is driedand etched with 37% phosphoric acid gel etchant (T.C gel)taking care not to contact pulp. After rinsing with distilledwater to remove the etchant, the cavity is dried but notdesiccated.

The exposed pulp is covered with the experimentalcapping materials, and light cured according to manufacturesinstruction. After this, the cavity is filled incrementallyavoiding undue pressure on pulp with Curex MicrohybrideLight Cure Composite (DPI) and cured.

Bleeding on placement of bonding agent was noticed withtwo cases in Group II.

When calcium hydroxide is used as pulp cappingmaterial, after etching the cavity, dycal is applied on thepulp avoiding undue pressure on pulp after which bondingagent (Gluma Comfort) is applied over dycal and the cavitywall, light cured, and cavity restored with curexMicrohybride Light Cure Composite (DPI).

Post Operative Procedure

All the patients were reviewed after 15 days and wereevaluated by inquiring about the post operative period andby clinical examinations which included the following :

1. Inquiring about any post operative sensitivity orspontaneous pain, pain during intake of cold or hot foodwhile chewing or eating

2. Clinical examination of filling material3. Clinical examination of the teeth:

a. Percussionb. Cold vitality testc. Mobility

4. Clinical examination of adjacent soft tissues fortenderness, swelling or sinus tract.

On post operative review, all the 10 patients had intactfilling on the treated teeth.

Among the 10 patients one patient experienced mildmomentary sensitivity to cold for all the three pulp cappedteeth.

Fig 1 Periphery of pulp capping where odontoblasts areobserved.

AO - active odontoblasts

Fig 2 Below pulp cappingC- Composite resin

P- Pulp showing mild inflammationD- Dentin

Fig 3 Periphery of pulp capping where activeodontoblasts are observed


69

Cold vitality test was found to be negative in 2 teeth in apatient (each one belonging to group I & group II). Thepostoperative period was uneventful for the patient and theteeth were not tender to percussion. During extraction, boththe teeth were found to have periapical granulomas.

After extraction, the teeth were immediately fixed in 10%buffered formalin, after cutting the root tip to expose pulpapically for penetration of fixative. The collected specimenswere transferred to biomedical technology wing at Sree ChitraTirunal Institute for Medical Science and Technology(SCTIMST) for histological analysis.

Processing and embedding of teeth in resin was doneaccording to W.H.O protocol.

Section Cutting of Resin Embedded Tissues

The teeth were sectioned to a size of 100µm along thelongitudinal axis with a High speed Precision saw (ISOMET2000) and ground to 50µm and polished with Variable SpeedGrinder - Polisher (ECOMET 3). The sections were fixed on aclean glass slide using cyanoacrylate glue and stained withToluidine blue for histological examination. The sectionswere observed under camera attached TrinocularFluorescence microscope ECLIPSE E - 600.

On histological examination, 4 sections were excludedfrom the study due to poor histological staining, artifactinterfering histological analysis or section representing asite distant from the pulp exposure. In the excluded sections,1 belonged to Group I & II each, and 2 belonged to Group III.

The remaining 26 sections were examined and includedin the study (9 specimens in Group I & II, 8 specimens inGroup III).

The specimens were primarily evaluated for the followingvariables:

1. Odontoblastic activity at the periphery of the pulpcapping site.

2. Degree of inflammation near the pulp capping site.3. Remaining pulp status.The specimens were also observed for:1. Presence of odontoblasts below and at the periphery

of pulp capping in contact with the material.2. Presence of microleakage.

3. Dentinal bridge formation.The observed primary variables were scored as following.I. Odontoblastic activity - in percentage (in a

standardized area of 250µm at the periphery of pulpcapping).

II. Inflammation near the exposure site-just below andperiphery of pulp capping.

0- No inflammation- No infiltration of inflammatorycells.

1- Mild inflammation- Infiltration by a few PMNL, roundcells, and / or macrophages.

2- Moderate inflammation- infiltrated by a moderatenumber of PMNL, round cells, and / or macrophages.

3- Severe inflammation- Massive infiltration of PMNLand / or round cells that obscured the pulp structure.

4- NecrosisIII. Reaction of Remaining pulp0- No inflammation1- Mild inflammation2- Moderate inflammation3- Severe inflammation4- Necrosis

ResultsTable I, II and III shows odontoblastic activity,

inflammatory response remaining pulp status andrespectively in percentages

Stedbond (Group I)

Odontoblastic activity was looked for in the nearestodontoblastic zone. Odontoblastic activity was taken in 8cases as 1 specimen showed no odontoblasts in theperiphery due to severe necrosis. Active odontoblasts wereobserved in all the 8 cases in varying percentage (average:19.461%). (Fig 1)

Below the pulp capping, mild inflammation withneutrophils and macrophages was seen in 5 cases (Fig 2)and moderate inflammation in 3 cases with numerous foamcells observed below the pulp capping in one case. Severeinflammation was not observed in any cases. Severe necrosiswas observed in one case. Dilated capillaries were observed

Fig 4 Below pulp cappingC- Composite resin

P-pulp showing necrosisD- Dentin

Fig 5 Periphery of pulp capping where activeodontoblasts are observedAO - active odontoblasts

Fig 6 Below pulp cappingCH-Calcium hydroxide (Dycal)

P-Pulp showing moderate inflammationD-Dentin

Comparative evaluation of odontoblastic activity following direct pulp capping with two

bonding agents and calcium hydroxide in humans-A histological analysis


70

Sreeja Sreedhar

in most of the cases.Normal pulp architecture is maintained in the central

and lower portions of pulp in 6 cases with 2 specimensshowing mild inflammation. Necrosis was observed in 1case which was associated with microleakage.

No dentine bridge was observed in any of the specimensin Group I. Fibroblasts were observed in the odontoblasticzone in the periphery near pulp capping in 1 case indicatinginitiation of healing.

Single Bond (Group II)

Focal loss of coronal portion of pulp was observed in allthe 9 cases of Group II with presence of no odontoblastsbelow and in the periphery in contact with the materials.

Odontoblastic activity was noted in 8 cases as 1 specimenshowed no odontoblasts in the periphery due to necrosis.Active odontoblasts were observed in all the 8 cases invarying percentages. (Average: 11.420%).(Fig 3)

Mild inflammation was observed in 1 case, and moderatechronic inflammation with macrophages and fewlymphocytes was observed in 2 cases. Severe chronicinflammation with macrophages and lymphocytes wereobserved in the remaining 3 cases. Necrosis was observednear pulp capping in 3 cases.(Fig 4)

Hemorrhage between the material and cavity wall wasobserved in 2 cases with moderate to severe inflammationnear the pulp capping.

But the remaining radicular pulp appeared normal in 7

cases with necrosis in one case. Mild inflammation andnecrosis was noticed in 1 specimen each.

No dentin bridge formation was observed in any of thenine cases.

Calcium Hydroxide (Dycal)(Group III)

Focal loss of pulp was observed in all the 8 cases withmaterial in direct contact with the pulp. Odontoblasts werefound to be absent below and at the periphery of the pulpcapping in contact with the material in 7 cases.

Odontoblastic activity was noted in 7 cases as 1 specimenshowed no odontoblasts in the periphery due to necrosis.Active odontoblasts were observed in all the 7 cases invarying percentage (average: 18.844%)(Fig 5)

Mild inflammation was observed near the pulp cappingin 4 cases and moderate inflammation in 3 cases(Fig 6).Severe chronic inflammation with macrophages andlymphocytes was observed in 1 case.

Remaining radicular pulp showed mild inflammation in3 cases and rest of the specimens showed normal appearingpulp.

No microleakage was observed in any of the cases.

Statistical analysis

Statistical analysis was done using the soft wareSTATISTICA (Statsoft Inc, USA). Pearson Chi-square test wasdone for the statistical analysis of degree of inflammationand remaining pulp status. Odontoblastic activity wasstatistically analysed using Analysis of Variance (ANOVA).

DiscussionBiocompatibility is the ability of a material to perform

with an appropriate host response in a specificapplication(8).

Presence of active odontoblasts in the periphery of thepulp capping is a sign of active synthesis of reparative dentin,which is an early sign of dentin bridge formation after pulpcapping(6, 7).

The type of adhesive system when used for direct pulpcapping is thought to induce different pulpal responses(9)and varying degrees of microleakage(10).

This study was conducted to examine the odontoblasticactivity and to assess the biocompatibility of two bondingagents (Stedbond and Single Bond) and also the immediatecellular events following their placement on mechanicallyexposed pulp compared with Dycal.

The pulp reparative process following direct pulp cappingis characterized by early neutrophilic response, a subsequentmacrophage response and a fibroblastic response that leadsto the deposition of reparative dentin or calcified-bridgeformation by odontoblast-like cells.

In our study, Single Bond showed severe pulpal responsecompared with other two materials. Severe inflammationand necrosis was observed in 33.33% cases(Table II). Thematerials can be ranked with respect to degree ofinflammation adjacent to the pulp capping site as: Single

Table: I Odontoblast activity in percentage

Specimen

1

2

3

4

5

6

7

8

Mean Percentage

Group I

17.14

13.95

20.75

20.83

26.086

22.41

24.39

10.14

19.461

II

11.53

10.79

8.16

15.58

8.19

6.25

16.98

13.88

11.420

III

10.44

21.42

16.66

29.41

22.53

19.67

11.76

18.844

The materials showed significant difference with respect to odontoblastic

activity.

SS

Effect

315.0603

df

Effect

2

MS

Effect

157.5301

SS

Error

563.2891

df

Error

20

MS

Error

28.16445

F

5.593225

P

0.011766


71

did not undergo apoptosis showed cell-cycle arrest.Few reports indicate that unpolymerized monomers may

be responsible for pulp inflammation and necrosis(14).Cytotoxicity of the dentin bonding components are ranked

as: Bis-GMA>UDMA>TEGDMA>>>HEMA (least toxic) after 24and 72-hour exposures. The three binary combinations ofdentin bonding components was found to interact in threeways: synergism, additivism, and antagonism - which wereinfluenced by the concentrations of both components(17).

Further studies have demonstrated an association ofTEGDMA - induced cytotoxicity with a rapid and drasticdepletion of cellular glutathione (GSH) followed by aproduction of reactive oxygen species (ROS)(18). A similarmechanism has been shown for HEMA-induced cytotoxicity,including cell cycle perturbation, and even apoptosis(19).

Both of the bonding agents used in the study containsHEMA as the primer but Stedbond uses acetone and SingleBond uses ethanol as the solvent. These solution will causedenaturation and precipitation of the proteins contained indentinal fluid. They can destroy cell and odontoblasticprocesses by extracting lipids from their membrances(7).

Recently, the incorporation of the antibacterial monomer,12-methacryloyloxydodecyl-pyridinium bromide (MDPB),has been shown to be effective in providing dentin primerswith antibacterial effects before and afterphotopolymerization. Nevertheless, more than 250g / mL ofMDPB need to be incorporated in the resin materials, suchas self-etching primers, to obtain effective antimicrobialactivity. At these concentrations MDPB is toxic to humancells(14).

Dentin adhesives have been shown to releasephotoinitiator camphoroquinone (CQ). It has beendocumented, not only as a cytotoxic agent, but also as amutagen. CQ incorporated into bonding agents could be, inpart responsible for the observed cytotoxicity(14).

However, there was no information available from the

Groups Mild SevereModerate Necrosis Row total

Group

I

II

III

All

groups

5 55.56

1 11.11

4 50.00

10 38.46

3 33.33

2 22.22

3 37.50

8 30.77

0 0.00

3 33.33

0 0.00

3 11.54

1 11.11

3 33.33

1 12.50

5 19.23

9 34.62

9 34.62

8 30.77

26

Pearson Chi-square: 10.1563, df=6, p=.118264There is no significant difference between the materials with respect to degree of inflammation near pulp capping.

Table: II Inflammation - in percentage

Bond> Dycal=Stedbond. But on statistical analysis, this wasfound to be insignificant, which may be due to inadequatesample size.

Schroder(11) noted that slighter the inflammation, higherwill be the frequency of bridging.

We also observed bleeding following placement of SingleBond on pulp in two cases in this study which was associatedwith severe inflammation and necrosis. This finding is inaccordance with that observed by Silva GA et al(12) in hisstudy on human dental pulp. Also Tasman F et al(13)demonstrated epinephrine -like contractions on the ratcarotid artery with Prime and Bond 2.1, Prime and Bond NT,and Syntac Single component except for Single Bond.

In our study, it was found that application of Single Bondproduced initiation of bleeding which interfered withrestorative procedure.

Hemorrhagic site have high oxygen tension which caninhibit polymerization of the bonding agents and can affectbonding(14). This can also be suggested as an additionalfactor for the severe inflammatory response observed withSingle Bond.

Ghavamnasiri et al (15) observed severe inflammationin Single Bond group even after 30 days.

In this study Single Bond also showed considerabledecrease (p=0.011766) in the odontoblastic activitycompared with other two materials which was found to bestatistically significant. This finding is in agreement withthe finding of Mantellini et al (2003).

Mantellini MG16 (2003) proved the cytotoxicity of SingleBond by demonstrating that Single Bond induced apoptosisof MDPC -23 (derived from dental pulp which retainfunctional characteristics of odontoblasts andmacrophages), OD-21 (undifferentiated cells derived dentalpulp), and macrophages. The proportion of apoptotic cellswas dependent on the degree of adhesive resinpolymerization. Most cells exposed to adhesive resin that




72

Sreeja Sreedhar

manufacturers on the percentage by weight or volume of themonomer constituents as well as other additives comprisinginorganic fillers, photo initiators and solvents.

The other material used in the present study wasStedbond. Minimal inflammation was observed with thismaterial. Severe inflammation was not observed in any ofthe cases except necrosis of the entire pulp in one specimenwhich was found to be associated with microleakage. Withthis material, an increased number of active odontoblastssecreating reparative dentin was observed (19.461%) whichis slightly higher and comparable with that observed withcontrol group-Dycal (18.844%) which suggests that thematerial is biocompatible (Table I). Also an initiation ofhealing was observed in one specimen of this group.

The appearance of macrophages 30 days postoperativelyhas been reported, when 4META / MMA-TBB resin is applieddirectly on the pulp. The appearance of macrophages couldresult from the irregular surface of the resin-pulp interface.This might be caused by some constituent dissolved fromthe resin(4). Numerous macrophages were visible in theaffected area of the pulp which was also observed with Dycalby Tronstad(20).

In the present study, macrophages and lymphocytes wereseen in 5 cases each with Stedbond, and Single Bond andonly in 1 case with Dycal group.

Dycal (a hard setting calcium hydroxide) is used as thecontrol group. With this material also minimal inflammationwas observed. Mild inflammation was observed with 50% ofthe cases, moderate inflammation in 37.50% cases. Severe

inflammation was observed in no cases. Necrosis wasobserved only in 12.50% cases(Table II).

Variable reaction of pulp to Dycal material has beenreported by Holland et al Kirk et al(21). But they could notfind the cause for this. Holland et al observed that it occurredindependently of alteration of the base-catalyst ratio. Thecomplex formula of Dycal cement may cause an inconsistentcalcium - release profile and the pulp will respond in one oftwo ways (either by producing a necrosis zone or withoutany necrosis), depending on whether the calcium level isabove or below a certain threshold value, which is as yetundetermined.

Scarano A et al(7) observed active odontoblasts at theperiphery of the capping in contact with material withdifferent adhesive resins and calcium hydroxidepreparations.

In the present study, few odontoblasts were observed incontact with material only in one case which belonged toControl (Dycal) group.

Although the materials varied in their pulp irritationpotential, all the materials maintained normal pulparchitecture in the remaining portion of the pulp( Table III).The case in which necrosis was found with Stedbond wasassociated with microleakage. This confirms the need toprevent microleakage for a successful pulp capping. No dentinbridge was observed with any of the materials in this periodof observation.

Summary & conclusion

Table: III Remaining pulp status

9 34.62

9 34.62

8 30.77

26

Groups Mild SevereModerate Necrosis Row total

I

II

III

All

groups

2 22.22

1 11.11

3 37.50

6 23.08

No

6 66.67

7 77.78

5 62.50

18 69.23

0

0

0

0

0

0

0

0

1 11.11

1 11.11

0 0.00

2 7.69

Pearson Chi-square: 2.32716, df=4, p=0675831There is no significant difference between the materials with respect to status of remaining pulp tissue.

Among the 3 materials studied Single Bond showed highest degree of pulpal inflammationcompared with other two materials. But statistically this difference was not significant due to inadequate sample size (p=0.118264).

All the materials showed odontoblastic activity near the pulp capping but it was significantlydecreased in Single Bond group (p=0.011766). Significant difference exists between Single Bond and Stedbond (p=0.022853) and also between Single Bond

and Dycal (p=0.044427). But no significant difference exists between Stedbond and Dycal (p=0.975118). Odontoblastic activity with Stedbond was comparable withthat of Dycal.

There was no statistically significant difference between the materials with respect to remaining pulp status (p=0.675831).


73

In summary, in the present study, compared to Stedbondand Dycal, Single Bond elicited more severe pulpal response(Severe inflammation and necrosis was observed in 33.33%cases each). It confirms that the inflammation of the pulpwith Stedbond and Dycal are reversible and dosen’t provokepulp necrosis.

Active odontoblasts were found to be significantly lessin number near the pulp capping with Single Bond comparedto other two materials.

Stedbond and Dycal showed minimal pulpal responseand seemed to have good tissue tolerance as they did notinduce severe tissue damage.

In conclusion, Stedbond and Dycal were found to benontoxic and biologically compatible to pulpal tissues whenplaced on mechanical pulp exposures after hemorrhagecontrol and as per manufacturer’s instructions. Single Bondcannot be considered biocompatible. Stedbond appearspromising as direct pulp capping agent. But it requires furtherlong term study to evaluate this bonding agent’s efficacy toform dentinal bridge and more research before it can beindicated as a direct pulp capping agent.

AcknowledgementThe authors wish to acknowledge the contribution of Dr.

Meera Mohanty, Head , Division of Pathophysiology ,SCTIMST- BMT, Trivandrum for her expert guidance in thisstudy.

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13. Tasman F, Cehredi ZC, Onur MA, Gumrukcuoglu A. Effect of different single-bottle dentin adhesives on vascular responses in rat carotid artery. Am J Dent.2000 Dec; 13 (6): 337-9.

14. Gurpinar A, Mehmet Ali Onur, Cehreli ZC, tasman F. Cytotoxicity of Two - stepSelf - etching Primer / Adhesives on L929 Cells. Journal of Bioactive andCompatible Polymers, 2006 Vol. 21, No. 1, 55-69.

15. Ghavamnsiri M, Maleknejad F, Salhenejad J, Moosavi H. A HistopathologicalStudy of Direct Pulp Capping with Adhesive Resin. J Dent TUMS 2004; Vol.1,No.4.

16. Mantellini M.G., T.M. Botero, P. Yaman, J.B. Dennison, C.T. Hanks, and J.E.Nor. Adhesive Resin Induces Apoptosis and Cell-cycle Arrest of Pulp Cells. J.Dent. Res 2003; 82 (8): 592-596.

17. Ratanasathien. S, J. C. Wataha, C. T. Hanks and J. B. Dennison Cytotoxicinteractive effects of dentin bonding components on mouse fibroblasts. Journalof Dental Research, 1995; Vol 74, 1602-1606.

18. Lefeuvre M, Bourd K, Loriot MA, Goldberg M, Beaune P, Perianin A, StanislawskiL. TEGDMA modulates glutathione transferase P1 activity in gingivalfibroblasts. J. Dent. Res. 2004; 83 (12): 914-990.

19. Schweikl H, Spagnuolo G, Schmalz G. Genetic and cellular toxicology of dentalresin monomers. J. Dent. Res. 2006; 85 (10): 870-877.

20. Tronstad L. Reaction of the exposed pulp to Dycal treatment. Oral Surg. Dec1974; 945-55.

21. Kirk EEJ. A comparison of dentinogenesis on pulp capping with calcium hydroxidein paste and cement form. Oral Surg Oral Med Oral Pathol Aug 1989; 68 (2):210-90.


74

irreversible pulpitis. The null hypothesis tested was thatthere is no significant difference amongst the three groups.

Materials and methodsNinety adult patients, who reported with pain to the

emergency dental department, within the age group of 20 –40 years were selected for the study.

Exclusion criteria were as follows: subjects who hadallergies, sensitivity, or were unable to take the drugs, wereallergic to local anesthetics or sulfites; were pregnant ornursing; had a history of significant medical conditions; orwere unable to give informed consent. The patients includedhad not taken any analgesics for at least 8 hours beforeenrollment in the study. To qualify for the study, each patienthad a vital mandibular first molar tooth, was activelyexperiencing pain, and had a prolonged response to coldtesting with Green Endo-Ice (1,1,1,2 tetrafluoroethane;Hygenic Corp, Akron, OH). Patients with no response to coldtesting, periradicular pathosis (other than a widenedperiodontal ligament), or no vital coronal pulp tissue onaccess were excluded from the study.

Ethical approval was sought from the Institutional ReviewBoard and Ethical committee of the University. Informedwritten consent was obtained from each subject.

The efficacy of pre-operative oral medication of Ibuprofen andTenoxicam on the success of inferior alveolar nerve block in patients withirreversible pulpitis: a double-blind, randomized controlled clinical trial

* Sulthan Ibrahim Raja Khan, * Ramachandran Anupama, * Mohanavelu Deepalakshmi, ** Kumarappan Senthil kumar,

* Senior Lecturers** Professor, Department of Conservative Dentistry & Endodontics, Chettinad

Dental College, Rajiv Gandhi salai, Kelambakkkam, Kanchipuram, India -6031 03

Aim: To determine the effect of administration of preoperative Ibuprofen (IBUP) or Tenoxicam (TENO) on thesuccess of inferior alveolar nerve blocks (IANB) in patients with irreversible pulpitis in a double blind randomisedcontrolled trial.

Methodology: Ninety patients with irreversible pulpitis of a mandibular first molar participated. Patientsindicated their pain scores on a Heft Parker visual analogue scale, after which they were randomly dividedinto three groups (n = 30). The subjects received identical capsules containing 800mg IBUP, 20 mg TENO orcellulose powder (placebo, PLAC), 1 h before administration of IANB with 2% lidocaine containing 1 : 200 000epinephrine. Access cavities were then prepared and success of IANB was defined as the absence of painduring access preparation and root canal instrumentation. The data were analysed using chi-squared tests.

Results: The success rates for the IANB in descending order were 72% (IBUP), 69% (TENO) and 26.5 %(PLAC). Asignificant (P < 0.001) difference was found between the drug groups and the PLAC group.

Conclusions: Pre-operative administration of IBUP & TENO significantly improved the efficacy of IANB inpatients with irreversible pulpitis.

Key words: Ibuprofen, Tenoxicam, inferior alveolar nerve block, irreversible pulpitis, local anaesthesia.

Abstract

Introduction

The inferior alveolar nerve block (IANB) is the mostcommonly used technique for achieving pulpal anesthesiafor mandibular teeth. IANB is highly effective in producinganaesthesia but also the most challenging because of itshigh failure rate in inflamed tissue [1]. The success rates ofIANB in cases of irreversible pulpitis, is reportedly as low as20%. [2] In irreversible pulpitis, there is an influx ofneutrophils and the release of inflammatory mediators suchas prostaglandins (PGs) and interleukins (ILs) andproinflammatory neuropeptides such as substance P,bradykinin, and calcitonin gene-related peptide. Thesemediators, in turn, sensitize the peripheral nociceptorswithin the pulp of the affected tooth, which increases painproduction and neuronal excitability which in turn causesreduced efficacy of anesthesia. [2, 3]

NSAIDs reversibly inhibit cyclooxygenase (prostaglandinendoperoxide synthase), the enzyme-mediating productionof prostaglandins (PGs) and thromboxane A2. Thus, byreducing the PGs one can hypothetically increase the efficacyof IANB.

Therefore, the purpose of this prospective, randomized,double-blind study was to compare the efficacy of oral pre-medication of Tenoxicam (TENO), ibuprofen (IBUP), and aplacebo (PLAC) medication on anaesthetic efficacy of IANBwith lidocaine with 1 : 200 000 epinephrine in patients with


75

Randomization of patients was done by simple randomsampling with a linear congruential generator. Randomizedallocation of the patients was done by a trained dentalhygienist who was blinded to the treatment procedures.

Every patient was asked to rate his/her pain on a HeftParker visual analogue scale (VAS) with 170-mm line markedwith various terms describing the levels of pain (Heft &Parker 1984). The millimetre marks were removed from thescale, and the scale was divided into four categories: nopain corresponded to 0 mm; faint, weak or mild paincorresponded to 1–54 mm; moderate to severe paincorresponded to 55–114 mm; and strong, intense, maximumpossible pain corresponded to more than 114 mm [4]

A dental hygienist divided 90 empty capsules of samecolor and size into 3 bottles: Tenoxicam (TENO), ibuprofen(IBUP) and placebo (PLAC) group. TENO capsules were filledwith 20 mg of Tenoxicam , IBUP capsules were filled with 800mg of ibuprofen, and PLAC capsules were filled with starch.The bottles were masked with an opaque label and wererandomly assigned a 3-digit alphanumeric value. A traineddental hygienist randomly divided all the patients into 3groups of 30 patients each and gave 1 capsule 1 hour beforethe procedure.

After 1 h of oral administration of the capsules, allpatients received standard IANB injections using 1.8 mL of2% lidocaine containing 1 : 200 000 epinephrine (Xylocaine;AstraZeneca Pharmaceutical Products, Wilmington, DE, USA).After reaching the target area, aspiration was performed,and the solution was deposited at a rate of 1 ml/ min by asingle operator.

After 15 minutes of the initial IANB, the involved teethwere isolated with a rubber dam, and a conventional accessopening was initiated. Patients were instructed to raise theirhand if any pain was felt during the procedure. In case ofpain during the treatment, the procedure was stopped, andpatients were asked to rate the pain on Heft Parker VAS. Theextent of access preparation and/or instrumentation was

recorded as within dentin, within pulpal space, andinstrumentation of canals. Success was defined as no painor weak/mild pain during endodontic access preparationand instrumentation.

The findings were recorded on a Microsoft Excel sheet(Microsoft Office Excel 2003; Microsoft Corp, Redmond, WA)for statistical evaluation by using the SPSS software.

Age and initial VAS scores were tabulated and comparedusing multiple comparison analysis of variance and posthoc tests. The genders of the patients were compared by chisquare test. The significance level was set at P = 0.05 forthese analyses. The proportions of successful IANB in the 3groups were compared using the chi-squared test.

Results:The age, gender and mean initial VAS scores were

tabulated (Table 1). There were no significant differences (P> 0.05) between the three groups. All patients reported asignificant decrease in active pain after local anaesthesia(P < 0.05). The post-injection VAS scores are given in Table 2.There was no significant difference between IBUP and TENOin the post-injection VAS scores (P > 0.05), whilst both IBUPand TENO resulted in significantly lower mean VAS scoresthan PLAC (P < 0.05). The percentage of patients withsuccessful IAN block are presented in Table 2.The percentageof successful IAN blocks was as following:26.5% in thecontrol PLAC group, whilst premedication with IBUP and TENOresulted in72% and 69% successful IANBs, respectively. Thepercentage of successful IANBs was significantly higher inthe IBUP and TENO groups when compared with the PLACgroup (P < 0.001).

Discussion:

The high failure rate of local anaesthesia in symptomaticteeth with irreversible pulpitis could be attributable to theprostaglandin-induced sensitization of peripheralnociceptors[5]. Prostaglandins (PGs) up-regulate a variety

ControlIbuprofen Tenoxicam(placebo)

Total number

of subjects 30 30 30

Age in years

(mean ± SD)* 27 ± 6 28 ± 4 30 ± 6

Gender*

Male 12 17 14

Female 18 13 16

Initial Pain (VAS)* 102 ± 18 99± 20 98 ± 21

*There were no significant differences between the three groups (P > 0.05).

TABLE - 1 : Comparison of age, gender and initial Heft Parkervisual analogue scale (VAS) scores amongst the three groups

Post-injection

Treatment IANB visual analogue

Group successful (%) scale scores

(Mean ± SD)

Control - Placebo 26.5% 20 ± 2a

Ibuprofen 72% 8 ± 2b

Tenoxicam 69% 7 ± 2b

Mean values that share a superscript letter were not significantly different atthe 5% level.

TABLE - 2 : Comparison of percentage of inferior alveolarTreatment group nerve block amongst the three groups

The efficacy of pre-operative oral medication of Ibuprofen and Tenoxicam on the success of inferior alveolar

nerve block in patients with irreversible pulpitis: a double-blind, randomized controlled clinical trial


76

of mechanisms that might decrease the efficacy of localanaesthetics: altering the kinetics of activity of the voltage-gated sodium channels, resulting in increaseddepolarization; activation of EG protein-coupled receptorsnamely P2 or EP3 receptors, which are expressed ontrigeminal sensory neurons [6] Several studies have shownthat the levels of prostaglandins are increased in inflameddental pulps [7]. Therefore, it can be concluded that byreducing the amount of prostaglandins, voltage gated sodiumchannels become less active thereby increasing theanaesthetic success.

The choice of tenoxicam and ibuprofen as premedicationsin this study came from the facts that these are relativelysafe, fast-acting analgesics that also control inflammation.The drugs studied could be clinically useful premedicationsbecause of their fast-acting, rapid release formulation andeffectiveness on dental pain. Premedication was given 1 hourbefore the procedure to allow NSAIDs to achieve satisfactoryplasma concentration.

Two percent lidocaine was chosen because severalstudies comparing lidocaine to other anesthetics, includingarticaine, in the success of pulpal anesthesia found little orno significant difference in efficacy. Mikesell et al. [8]concluded that 4% articaine with 1:100,000 epinephrine wassimilar to 2% lidocaine with 1:100,000 epinephrine in inferioralveolar nerve blocks. Claffey et al[9] found the IAN successrate for patients with irreversible pulpitis receiving articainewas 24% and for the lidocaine solution success was 23%.Tofoli et al. [10] found 4% articaine with 1:100,000 or1:200,000 epinephrine equally effective for IAN block.

The results of the study show that the increase inanaesthetic effect of lidocaine was statistically significantin both the two drug groups compared to the placebo group.Similar results were found by Ianiro et al [11] withacetaminophen and ibuprofen.

While ibuprofen and tenoxicam are both NSAIDs, theydo have different chemical structures. Both inhibit the cycle-oxygenase enzyme system and hence reduce the biosynthesesof prostaglandins. Ibuprofen blocks COX-1 and COX-2enzymes. It is safe, widely prescribed, inexpensive and haseffective analgesic and anti-inflammatory action for post-operative pain. [12] In addition, ibuprofen also inhibits themigration and other functions of leucocytes.

Tenoxicam is rather frequently prescribed because itspecifically inhibits COX-2, which could imply a lowerincidence of undesirable side effects such as gastricintolerance (13,14). By preventing the formation of PGs, iteffectively controls inflammation. A few studies in oralsurgery models (15,16,17) and orthodontics (18) havecompared tenoxicam and other NSAIDs. Tenoxicamadministered orally or parenterally is an effective analgesicand anti-inflammatory agent for the symptomatic treatmentof various conditions, including rheumatoid arthritis and

osteoarthritis. Also, it is characterized by lower penetrationinto tissues, which explains reduced incidence of adversereactions owing to this drug.[19]

Thus, it can be seen that prostaglandins are effectivelyblocked by both IBUP and TENO. From the results of thepresent study, it may be speculated that these NSAIDs can begiven as a premedication to increase the efficacy of IANB.

Conclusion:Oral pre-medication with 800 mg IBUP and 20 mg TENO

resulted in significantly higher percentage of successful IANBanaesthesia in patients with irreversible pulpitis than pre-medication with PLAC.

References:1. Nusstein JM, Reader A, Drum M. Local Anesthesia Strategies for the Patient

with a ‘‘Hot’’ Tooth. Dent Clin N Am 54 (2010) 237–2472. Hargreaves K, Keiser K. Local anesthetic failures in endodontics: mechanism

and Management (a review). Endod Topics. 2002;26-39.3. Dray A. Inflammatory mediators of pain. Br J Anaesth 1995;75(2):125–31.4. Heft MW, Parker SR. An experimental basis for revising the graphic rating scale

for pain. Pain 1984;19:153–61.5. Henry MA, Hargreaves KM (2007) Peripheral mechanisms of odontogenic

pain. Dental Clinics of North America 51, 19–44.6. Vane JR, Botting RM (1998) Mechanism of action of nonsteroidal anti-

inflammatory drugs. American Journal of Medicine 104, 2S.7. Ingle B, Baumgartner. Ingle’s endodontics. 6th ed. Hamilton, Ontario, Canada:

BC Decker Inc; 20088. Mikesell P, Nusstein J, Reader A, Beck M, Weaver J. A comparison of articaine

and lidocaine for inferior alveolar nerve blocks. J Endod 2005;31:265–70.9. Claffey E, Reader A, Nusstein J, Beck M, Weaver J. Anesthetic efficacy of

articaine for inferior alveolar nerve blocks in patients with irreversible pulpitis.J Endod 2004; 30:568.

10. Tofoli GR, Ramacciato JC, de Oliveira PC, Volpato MC, Groppo FC, Ranali J.Comparison of effectiveness of 4% articaine associated with 1:100,000 or1:200,000 epinephrine in inferior alveolar nerve block. Anesth Prog2003;50:164–8.

11. Ianiro SR et al. The effect of preoperative acetaminophen or a combination ofacetaminophen and ibuprofen on the success of inferior alveolar nerve blockfor teeth with irreversible pulpitis. J Endod 2007;33:11–14)

12. Richy F, Bruyere O, Ethgen O, Rabenda V, Bouvenot G, Audran M, Herrero-Beaumont G, Moore A, Eliakim R, Haim M, Reginster JY (2004) Time dependentrisk of gastrointestinal complications induced by non-steroidal anti-inflammatory drug use: a consensus statement using a meta-analyticapproach. Ann Rheum Dis 63, 759-766.

13. Ezberci F, Bulbuloglu E, Ciragil P, Gul M, Kurutas EB, Bozkurt S, Kale IT (2006)Intraperitoneal tenoxicam to prevent abdominal adhesion formation in a ratperitonitis model. Surg Today 36, 361-366.

14. Van Antwerpen P, Nève J (2004) In vitro comparative assessment of thescavenging activity against three reactive oxygen species of non-steroidal anti-inflammatory drugs from the oxicam and sulfoanilide families. Eur J Pharmacol496, 55-61.

15. Cheung LK, Rodrigo C (1992) Tenoxicam for pain relief following third molarsurgery. Anesth Pain Control Dent 1, 229-233.

16. Roelofse JA, Swart LC, Stander IA (1996) An observer-blind randomised parallelgroup study comparing the efficacy and tolerability of tenoxicam and piroxicamin the treatment of post-operative pain after oral surgery. J Dent Assoc S Afr 51,707-711.

17. Uçok C (1997) Stereophotogrammetric assessment of the effect of tenoxicamon facial swelling subsequent to third molar surgery. Int J Oral Maxillofac Surg26, 380-382.

18. Arantes GM, Arantes VM, Ashmawi HA, Posso IP (2009) Tenoxicam controlspain without altering orthodontic movement of maxillary canines. OrthodCraniofac Res 12, 14-19.

19. Starek M, Krzek J (2009) A review of analytical techniques for determinationof oxicams, nimesulide and nabumetone. Talanta 77, 925-942.

Sulthan Ibrahim Raja Khan


77

Fluoride Enhanced Hydroxyapatite Gel (Remin Pro,Voco)combines three components, Hydroxylapatite, Fluoride &Xylitol. It is claimed to be an effective protection againstdemineralisation and erosion with the hydroxyapatite inRemin Pro promoting remineralisation as well asdesensitisation of the tooth substance. The cream is waterbased with a pleasant flavour. Remin Pro adheres to thetooth surface, smoothening the tooth surface and sealsdentinal tubules superficially as per the manufacturer. Thesugar substitute xylitol present is known for its cariostaticproperties.

Propolis is a resinous wax-like material that is used bythe bees as a glue-like matrix in their hives. It has beenreported to bear antibacterial activity that may be of benefitin combating dental caries.Studies on Propolis applicationshave increased because of its therapeutic and biologicalproperties(4).

Of all the techniques used in dentistry to enhanceenamel’s resistance to acid attack, fluoride application andCO

2 laser have shown a satisfactory outcome. Laser

technology was first used in dentistry for the removal ofcaries infected material and preparation of cavities.Attention has been focused on the laser’s potential toenhance enamel’s hardness and resistance to acid(5).Different types of lasers, such as Ruby, Carbon Dioxide,Neodymium:Yttrium-Aluminum-Garnet, and Argon, withdifferent operational modes and energy outputs, have been

Multiple irradiation of enamel surface in conjunction with acidulatedphosphate fluoride (APF) gel, fluoride enhanced hydroxyapatite gel

(Remin Pro) and propolis-A SEM evaluation* Mathew Varghese K, ** Ajit George Mohan

* Reader,** Senior Lecturer, Department of Conservative Dentistry & Endodontics, Annoor

Dental College, Muvattupuza, Kerala

Aim: To investigate the surface changes on enamel when exposed to multiple CO2 irradiation in conjunction

with topical remineralising agents.

Materials & Method: Extracted intact 40 human maxillary central incisors were collected and stored in saline.Roots were resected and the crowns were washed in distilled water and stored in saline at room temperature.Group I consisted of untreated enamel surface which served as the control. Group II reflected the applicationof APF gel, Group III involved the application of Fluoride enhanced hydroxyapatite gel and in Group IV propolisapplication was done. Laser irradiation was carried out before after the application of the topical gels.

Results: All the samples presented with crater formation on the surface. CO2 irradiation along with fluoride

enhanced hydroxyapatite gel revealed a surface with fewer craters.

Conclusion: Multiple CO2 laser irradiation of enamel through a topically applied remineralising gel can be

useful and effective in the prophylaxis and management of patients at risk for dental caries.

Abstract

IntroductionThe urge and search for new methods for the prevention

of dental caries by increasing the acid resistance capacityof the enamel is a very noble way in preventing tooth decay.Many attempts have been made to alter tooth enamel toincrease its resistance to caries. If it were possible toeliminate the pits and fissures from the enamel surface byfusing or “welding” the enamel, the susceptibility of teeth tocaries might be considerably decreased. With the use oftechnological advancements like lasers in dentistry, thescope for prevention of caries has increased multifold.

Over time, modern dentistry has evolved to a minimallyinvasive approach in which, caries is managed as aninfectious disease, deferring operative intervention as longas possible. The focus is on maximum conservation ofdemineralized, noncavitated enamel and dentin(1). Enamelis the strongest, most esthetic and resistant material thatneeds to be retained as far as possible. It is vital to attemptarrest of active lesions and facilitate remineralisation ofinitial lesions prior to attempting any surgicalintervention.(2)

The anticariogenic effect of professional fluorideapplication depends on reaction products formed on enamelduring the clinical treatment and their retention over timeafter the application. The formation and retention of fluoridecompounds on enamel is dependent on the concentration offluoride applied and the pH of the commercial product used.Professional acidulated phosphate fluoride (APF)application is a well-known method used for dental cariesprevention and its efficacy is clearly recognized on anevidence-based perspective(3).


78

Mathew Varghese K.

used to investigate the possibility of dental cariesprevention.(6-17)

Even though the resistance of enamel to acid attack byirradiation with CO

2 laser and fluoride application has been

reported, the effect of a natural resin on the enamel incombination with CO

2 laser was never reported. Hence the

aim of the present study is to investigate the surface changeson enamel when exposed to multiple CO

2 irradiations in

conjunction with topical remineralising agents.

Materials & methods

Extracted intact 40 human maxillary central incisorswere collected and stored in saline. Roots were resected andthe crowns were washed in distilled water and stored insaline at room temperature. The palatal surface of the crownswas flattened using an acrylic trimmer. The 40 crowns wererandomly divided into 4 groups. Nail varnish was appliedsuch that only the labial surface was exposed. Group I wasthe untreated enamel surface which served as the control. InGroup II APF gel was applied, Group III involved theapplication of Fluoride enhanced hydroxylapatite gel andin Group IV propolis application was done. Laser irradiationwas carried out before and after the application of the topicalgels.

Group I = Control (n=10),Group II = CO

2 Laser irradiation before & after the

application of APF (n=10),Group III= CO

2 Laser irradiation before & after the

application of Fluoride enhanced hydroxylapatite gel(ReminPro) (n=10), (Mesio-incisal half)

Group IV = CO2 Laser irradiation before & after theapplication of Propolis (n=10).

Fluoride application technique

A single application of APF gel (Pascal Company, 2929N.E. Northrup Way, Bellevue, WA 98004, U.S.A), pH 3.5 ,wasperformed on the labial surface of the specimen using amicro-brush for 1 min.

Propolis application technique

The application was carried out in the same method asthat of fluoride application.

Remin-pro application technique

After 1 minute application of Remin-Pro using a brush,on the labial surface, the samples were immersed in artificialsaliva for 3 minutes as per the manufacturer’s instructions.

Laser irradiation techniqueCO

2 laser (Sunny Surgical Laser, model number-PC015C;

Shanghai, China) irradiation was carried out by a pulsedCO

2 laser at 10.6µm wavelength with the following

parameters: 0.5 W, 50-µs pulse duration, 1-Hz repetition rateand a 0.8mm beam diameter. A device made with orthodonticwire was fixed to the laser tip such that a distance of 4mmfrom the tip of the hand piece to the specimen was maintainedduring irradiation. The specimens were exposed forapproximately 15s by moving the laser tip manually.Necessary precautionary measures were taken by theoperator during the laser irradiation procedure.

The surface morphology of the test samples wereanalyzed using a High Resolution Scanning ElectronMicroscope (HR-SEM) (F E I Quanta FEG 200 - High ResolutionScanning Electron Microscope, FEI Company, North AmericaNanoPort, 5350 NE Dawson Creek Drive, Hillsboro, Oregon97124 USA). All the specimens were stored in artificial saliva.

The composition of artificial saliva used was1. Na3PO4 – 3.90 mM2. NaCl – 4.29 mM3. KCl – 17.98 mM4. CaCl

2 – 1.10mM

5. MgCl2 – 0.08 mM

6. H2SO

4 – 0.50 mM

7. NaHCO3 – 3.27 mM, distilled water, and the pH was

set at a level of 7.2.

Fig 1 – Control(Group I)Fig 2- APF gel with multiple irradiation of CO2(Group II)


79

ResultsFormation of craters was noted in the entire multiple

irradiated groups except for the control group where laserirradiation was not done. Multiple laser irradiations alongwith APF presented craters with incomplete closure of theenamel prisms whereas when a fluoride enhancedhydroxylapatite gel was used with multiple laserirradiations, a smoother surface was present with depositsof calcium fluoride on the surface. Mineral deposits werealso seen in group were enamel was irradiated along withpropolis.

DiscussionHard tissue modification by means of laser irradiation

is becoming popular in dentistry(18). Efforts have specificallybeen concentrated on new laser-based biomedical techniquesfor painless, contact-free, hard dental tissue interventions(19). Laser has been also proposed for preventive treatment,diagnosis of caries, etching of enamel, caries removal,debond ceramic brackets(20) and more recently, to enlargethe root canal during endodontic therapy(19). Attentionshould be paid to several issues concerning safety andefficacy of the lasing procedures. Indeed, the laser beam hasthe potential to penetrate through the tooth tissues and toaffect the underlying pulpal tissue as well. Both thermal andmechanical effects of the interaction are definitely influencedby the nature of the tissue target and the laser pulsecharacteristics(21). In the literature there are conflictingevidences on the effects of laser treatment on enamelstructure; this is most probably due to the high number ofvariables involved in the lasing process: power, pulsefrequency and duration of irradiation(22). Therefore, thechoice of the laser parameters for various applications iscrucial.

Fluoride has been found to be the most effectivecariostatic agent in the field of dentistry. In the past fewdecades it has completely changed the approach of treatmentfrom a therapeutic concept to a more preventive approach.

On the tooth it alters the physiochemical properties bymaking it more resistant to acid dissolution due to formationof fluorapatite or fluorhydroxyapatite. It also increases thepost-eruptive maturation, enhances remineralisation andinhibits demineralization(23). In the bacteria it inhibitsvarious enzymes like enolases, phosphatases, protonextruding ATPases and pyrophosphatases(24). Fluoride alsoinfluences the bacterial composition and alters the plaqueecosystem(25).

Due to the limited absorption coefficient (Â1 cm–1) ofenamel for the Nd-YAG laser(26), its 1.06- µm wavelength isnot absorbed effectively by human enamel. So the enamelsurface needs to be coated with a black ink to enhanceabsorption(27). The CO

2 laser, with an emission wavelength

of 10.6 µm, very close to the phosphate and carbonateabsorption bands of dental enamel apatite, is absorbed moreefficiently by dental enamel(28), causing a loss of carbonateand a reduction of reactivity at a sufficiently hightemperature(28,29). We therefore postulate that thetransformation of light to heat when absorbed into theenamel mineral is more effective at a wavelength of 10.6 µmthan at a 1.06-µm wavelength with a black mediator.

Marquez et al in 1993 explained that 30% of thecarbonate is lost between 400°C and 600°C and it iscompletely removed only after repeated irradiation beyondthe melting temperature of the enamel itself (more than800°C). The complete removal of the carbonate derives bothfrom the absorption depth of the surface and from pulseintensity and duration.(30) So a multiple irradiation beforeand after the application of topical gels might have led tothe complete removal of carbonate, thereby incorporatingthe mineral into the enamel. Phan et al. proposed themechanism for FAP transformation as follows: During thefluoride gel treatment, fluoride ions diffuse through the poresbetween the enamel rods to deposit and form a Fluoride-veneer layer covering all the enamel rods. Following the CO

2laser irradiation, this thin Fluoride-veneer layer along withthe superficial microlayer of enamel surface was thermally

Fig 3-Propolis with multiple irradiation of CO2 (Group III) Fig 4-Remin-pro with multiple irradiation of CO2 (Group IV)

Multiple irradiation of enamel surface in conjunction with acidulated phosphate fluoride (APF)

gel, fluoride enhanced hydroxyapatite gel (Remin Pro) and propolis-A SEM evaluation


80

melted, resulting in re-crystallization and rearrangement ofa new FAP mineral.(31) The CO

2 laser, with an emission

wavelength of 10.6 µm which is very close to the phosphateand carbonate absorption bands of dental enamel apatite,is absorbed more efficiently by dental enamel causing aloss of carbonate and a reduction of reactivity at a sufficientlyhigh temperature. (27-28) Effects of laser irradiation on thepulp were not investigated in the present study; previousstudies however suggested that the irradiation conditionsapplied here are safe. In the current investigation thefollowing settings were used: laser light wavelength of 10.6µm at an output of 0.5 W with an irradiation distance of 4mm to the sample and an irradiation time of 15 s. Anic andco-workers (1992) demonstrated that the CO

2 laser at 4 W

continuous wave for 15 s caused a temperature raise of 3.5–4.1 ºC in the pulp space. (32, 33)

There was presence of craters in the entire multipleirradiated groups except for the control where laserirradiation was not done. Multiple laser irradiations alongwith APF presented craters with incomplete closure of theenamel prisms. But when a fluoride enhancedhydroxylapatite gel was used with multiple laser irradiationsa smoother surface was present with deposits of calciumfluoride on the surface. Mineral deposits were also seen ingroup were enamel was irradiated along with propolissuggesting presence of minerals that might enhanceremineralisation. Giamalia et al reported an increase inmicrohardness of enamel when exposed to propolis.(4)

The current study investigated the beneficial effects whenenamel surfaces were wetted with a topical remineralisingagent before and after they were laser-irradiated. The use offluorides is one of the most effective methods in cariesprophylaxis. Fluoride helps control decay by enhancingremineralization and altering the structure of the tooth,making the surface less soluble. The anticariogenic effect ofprofessional fluoride application depends on reactionproducts formed on enamel during the clinical treatmentand their retention over time after the application. Lasertreatment in combination with fluoride application appearsto have several advantages over fluoride application alone.Following the CO

2 laser irradiation, the Fluoride-veneer layer

on the surface along with the superficial microlayer ofenamel surface was thermally melted, resulting in re-crystallization and rearrangement of a new FAP mineral. Thisfirmly bound fluoride minimizes the mineral loss from theenamel surface thereby making it more resistant to acidattack.

Multiple CO2 laser irradiation of enamel through a

topically applied remineralising gel may thus be useful andeffective in the prophylaxis and management of patients atrisk for dental caries.

References1. Carol Anne Murdoch-Kinch, Minimally invasive dentistry, JADA, Vol. 134,

January 2003.2. L Narayanan, CVN Rao. Future Advances in Conservative dentistry and

endodontics.1st edition, Meenakshiammal educational trust.3. Stern RH, Vahl J, Sognnaes RF: Laser enamel: Ultrastructural observations of

pulsed carbon dioxide laser effects. J Dent Res 1972; 51:455– 460.

4. I. Giamalia, D. Steinberg, The effect of propolis exposure on microhardness ofhuman enamel in vitro, Journal of Oral Rehabilitation 1999 26; 941–943.

5. Weimin GUAN, Nobuya HAYASHI, Satoshi IHARA, Reports of the Faculty ofScience and Engineering, Saga University, 2004, 33, 1.

6. Stern RH, Sognnaes RF, Goodman F: Laser effect on in vitro enamel permeabilityand solubility. J Am Dent Assoc 1966; 73:838–843.

7. Yamamoto H, Ooya K: Potential of yttrium-aluminum- garnet laser in cariesprevention. J Oral Pathol 1974; 3:7–15.

8. Yamamoto H, Sato K: Prevention of dental caries by Nd:YAG laser irradiation.J Dent Res 1980: 59:2171–2177.

9. Nelson DGA, Shariati M, Glena R, Shields CP, Featherstone JDB: Effect of pulsedlow-energy infrared laser irradiation on artificial carieslike lesion formation.Caries Res 1986:20:289– 299.

10. Nelson DGA, Wefel JS, Jongebloed WL, Featherstone JDB: Morphology,histology and crystallography of human dental enamel treated with pulsedlow-energy infrared laser radiation. Caries Res 1987:21:411–426.

11. Tagomori S, Morioka T: Combined effects of laser and fluoride on acid resistanceof human dental enamel. Caries Res 1989:23:225–231.

12. Oho T, Morioka T: A possible mechanism of acquired acid resistance of humandental enamel by laser irradiation. Caries Res 1990:24:86–92.

13. Fox JL, Yu D, Otsuka M, Higuchi WI, Wong J, Powell G: Combined effects oflaser irradiation and chemical inhibitors on the dissolution of dental enamel.Caries Res 1992:26:333–339.

14. Kumazaki M, Morimoto T, Tsuchihashi Y, Morikawa M, Arai T, Ueda Y: Studieson enamel solubility and acid resistance by laser. J Jpn Soc Laser Dent 1993:4:85–93.

15. Hicks MJ, Flaitz CM, Westerman GH, Berg JH, Blankenau R L, Powell GL: Caries-like lesion initiation and progression in sound enamel following argon laserirradiation: An in vitro study. J Dent Child 1993:60:201–206.

16. Westerman GH, Hicks MJ, Flaitz CM, Blankenau RJ, Powell GL, Berg JH: Argonlaser irradiation in root surface caries: In vitro study examines laser effects.JADA 1994:125:401–407.

17. Westerman GH, Hicks MJ, Flaitz CM, Powell GL, Blankenau RJ: Surfacemorphology of sound enamel after argon laser irradiation: An in vitro scanningelectron microscopic study. J Clin Pediatr Dent 1996:21:55–59.

18. Franke M, Taylor AW, Lago A, Fredel MC. Influence of Nd: YAG laser irradiationon an adhesive restorative procedure. Oper Dent 2006; 31:604-9.

19. Khabbaz MG, Makropoulou MI, Serafetinides AA, Papadopoulos D,Papagiakoumou E. Q-Switched vs. Freerunning Er:YAG laser efficacy on theroot canal walls of human teeth: a SEM study. J Endod 2004; 30:585-8.

20. Hayakawa K. Nd:YAG laser for debonding ceramic orthodontic brackets. Am JOrthod Dentofacial Orthop 2005; 128:638- 47.

21. Dayem RN. Evaluation of the ablation efficacy and morphology of some hardtissues irradiated with different types and modes of laser. Lasers Med Sci Oct2007.

22. Castellan CS, Luiz AC, Bezinelli LM, Lopes RM, Mendes FM, De P Eduardo C,De Freitas PM. In vitro evaluation of enamel demineralization after Er:YAG andNd:YAG laser irradiation on primary teeth. Photomed Laser Surg 2007; 25:85-90.

23. Bowden GHW. Microbiology of root surface caries in humans. J Dent Res 1990;69: 1205-1210.

24. Fejerskov V, Ekstrand J, Burt BA. Fluoride in Dentistry. 2nd ed. Munksgaard:Copenhagen; 1996: 311-327.

25. Hamilton IR. Biochemical effects of fluoride on oral bacteria. J Dent Res 1990;69: 660-667.

26. Fried D, Borzillary SF, McCormack SM, Clena RE,Featherstone JDB, Seka W:The thermal effects on CO2 laser irradiated dental enamel at 9.3, 9.6, 10.3 and10.6 Ìm; in Laser Surgery: Advanced Characterization, Therapeutics, andSystems IV. Bellingham, SPIE, 1994, pp 319–328.

27. Morioka T, Suzuki K, Tagomori S: Effect of beam absorptive mediators on anacid resistance of surface enamel by Nd-YAG laser irradiation. J Dent Health1984; 34:40–44.

28. Featherstone JDB, Nelson DGA: Laser effects on dental hard tissue. Adv DentRes 1987; 1:21–26.

29. Featherstone JDB, Fried D, Bitten ER: Mechanism of laser-induced solubilityreduction of dental enamel; in Lasers in Dentistry III. Bellingham, SPIE, 1997,vol 2973, pp112–116.

30. Marquez F, Quintana E, Roca I, Salgado J. Physical-mechanical effects of Nd:YAGlaser on the surface of sound dental enamel. Biomaterials 1993; 14:313-6.

31. Phan ND, Fried D, Featherstone JDB. Laser-induced transformation ofcarbonated apatite to fluorapatite on bovine enamel. In: Laser in dentistry. V.Bellingham: SPIE,1999. Proceedings of SPIE.1999; 3593:233-40.

32. Anic I, Vidovic D, Luic M, Tudja M. Laser induced molar tooth pulp chambertemperature changes. Caries Research 1992; 26:165–9.

33. YuD, PowellGL,HiguchiWI, Fox JL.Comparison of three lasers on dental pulpchamber temperature change. Journal of Clinical Lasers Medicine and Surgery1993; 11:119-22.

Mathew Varghese K.


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IntroductionThe concept of bonding a restorative material to dentinal

surface is by no means a new idea. Even at the time ofBuonocore using phosphoric acid to bond Enamel, the ideaof bonding to dentin was considered(1). The dentin bondingsystems have been developed over the years and have becomeof particular interest to the success of tooth coloredrestorative materials that include ceramics, composites andcompomers. With the introduction of newer generationbonding agents, the problem of technique sensitivity hasreduced(2).

The past decade has seen increased use of bonding agentsin conjunction with traditional dental materials. Patientswith severe deformities like dental fluorosis which wereearlier treated with full coverage crowns is seldom treatedwith such extensive treatment. Availability of adhesivetechniques have made use of bonded veneers, a treatment ofchoice for such defects. Veneers which are brittle, oncebonded to the tooth become stronger and clinically functional(3,4).

The successful use of dental adhesive materials relieson their proper selection and on understanding theirproperties, in particular their curing mechanism andapplication technique.

Enamel adhesion by means of acid etching has becomean accepted technique in restorative dentistry. Adhesion todentin, however is still under investigation. At this time, two

The aim of this study was to evaluate the bond strength of two newer generation simplified bondingsystems in comparison to conventional three step bonding system.

36 freshly extracted non carious intact human maxillary and mandibular teeth were selected for this study.They were divided into 3 groups of 12 teeth each. The occlusal surface of the teeth was ground to prepare flatdentin surface. In group I, 5th generation dentin bonding system Excite, (3 step bonding system)was used andin group 2, Prompt-L-pop, a self etching primer( all in one system) was used and in group 3, clearfil Liner bond2V( 2 bottle system) was used.. All three dentin bonding agents were placed on the flat dentinal surfacesaccording to the manufacturer’s instructions. Cylinders of composite resin were built on the prepared surfaceusing Teflon mould. The prepared specimen were then embedded on an acrylic jig of suitable dimension andtransferred to Instron machine for shear bond strength analysis. Shear bond strengths were measured at across head speed of 0.03mm/sec.

Key words: Total etch adhesive, Self etching Primer, Shear Bond, Excite, Prompt -L- Pop

Abstract

Comparative evaluation of the shear bond strength of conventionaltotal etch adhesive with simplified bonding systems

* Shamina Bawa, ** Mithra N.Hegde

* Reader, Dept. of Conservative dentistry and Endodontics, D.A.P.M.R.V. DentalCollege, J.P.Nagar, Bangalore-78;

** Prof & HOD, Dept. of Conservative & Endodontics, A.B.Shetty Memorial Instituteof Dental Science, Mangalore.

distinct adhesive techniques are prominent in the attempt toestablish a strong bond to dentin: Total etch and self etchingprimers(5).

Total etch adhesives have been shown to be effectiveboth in vitro and in clinical evaluation. Self etching primersare still undergoing rapid evolution. Though some systemshave achieved positive results, further studies are necessaryto confirm the long term efficiency of self etching primers.The aim of the present study was to evaluate and comparethe bonding efficiency of two different self etching bondingsystem with a total etch adhesive .

Materials and methods36 freshly extracted non carious intact human maxillary

and mandibular teeth were selected for this study. Theocclusal surfaces of the teeth were ground on a water cooledtrimming wheel to prepare flat dentin surface. They weredivided into 3 groups of 12 teeth each. Group 1 was bondedusing conventional three step total etch adhesive namelyExcite. Group II was bonded using one step self etching primernamely Prompt –L- Pop, and group III, two step self etchingprimer Clearfil Liner Bond 2V was used.

In group I, where Excite was used, the prepared flatdentinal surface was etched using 35% phosphoric acid for15 seconds. It was then cleansed and dried using oil free


82

water spray. Dentin bonding agent was then applied andcured according to the manufacturer’s instruction. This wasfollowed by packing of composite resin over the prepareddentinal surface using Teflon mould measuring 3X3 mm andcured layer by layer.

In group II, one step self etching primer Prompt-L-Popwas used. Here the liquid from the red blister was transferredinto the yellow blister which was then transferred into thegreen blister which has the applicator tip. Using thisapplicator tip, the bonding agent was applied to the prepareddentinal surface with moderate finger pressure.stream ofair was used to evenly disperse the material into thin film.The material was then cured for 20 seconds. Composite resinwas packed over this prepared surface using Teflon mouldand cured layer by layer.

In group III, where Clearfil Liner Bond 2 V was used, primerliquid A and B were mixed and then applied to the prepareddentinal Surface. It was dried with mild air stream and leftfor 30 seconds. Primer was not washed. Bonding agent wasapplied and light cured for 20 seconds. After curing thebonding agent, composite resin was packed using Teflonmould and cured according to manufacturer’s instruction.

The prepared specimen were then embedded on anacrylic jig of suitable dimension &transferred to Instronmachine for shear bond strength analysis. Shear bondstrengths were measured at a cross head speed of 0.03mm/sec.

ResultsThe present in vitro investigation evaluated the shear

bond strength of composite to dentin restored in conjunctionwith Excite, a total etch adhesive in comparison with theself etching primer Prompt –L- Pop, and Clearfil Liner Bond2V primer.

According to the results analyzed, the highest mean shearstrength value was produced by Clearfil Liner Bond 2V (19.53MPa),followed by Excite (14.7 MPa) and Prompt-L-Pop([13.55Mpa).

Intercomparision among groups was done usingBoneferonni‘t’ test. In group I &II comparison between bondstrength values of Excite & Prompt-L-Pop was not significant.The comparison between the bond strength values of Excite& Clearfil Liner Bond 2V was very highly significant. Veryhighly significant results were observed when Clearfil LinerBond 2V was compared with Prompt –L- Pop.

Discussion

Resins as restorative material is nothing new. However,clinicians have hesitated using 4th and 5th generation dentinbonding systems basically because of the time involved andthe number of steps involved in the procedure. The techniquesensitiveness in utilizing these materials also have reducedthe use of these materials in day to day practice. Becausebonding procedures require multiple step clinicalapproaches, clinical success with these adhesive systemssometimes depend on technique sensitive and materialrelated factors (5, 6). In an effort to simplify the bondingprocedure, several new adhesive systems rely onsimultaneous etching of enamel and dentin with phosphoricacid or self etching primers. The self etching primer systemscombine the tooth surface etching and priming steps to treatenamel and dentin simultaneously. The self etching primerswhich dissolve the smear layer and demineralise the dentinis an aqueous mixture of acidic functional monomers withother constituents(7).

Although the most reliable conclusion about theperformance of dentin bonding system in the oralenvironment must be derived from the long term clinicaltrials, laboratory tests are still necessary to evaluate thedifferent dentin bonding systems.

In this present investigation, we have made an attemptto evaluate a globally accepted total etch adhesive namelyExcite with self etching primer system,Clearfil Liner Bond 2Vand the more recently introduced self etching primer PromptL Pop.

Total etch adhesives involve a separate etch and rinsephase. The underlying mechanism of adhesion to dentin isalike for the three and two step total etch adhesives. Thedentin smear layer produced during cavity preparation isremoved by the etch and rinse phase, which concurrentlyresults in a 3-5micron deep demineralization of the dentinsurface8.

Self etching adhesives are based on the use of non rinseacidic monomers that simultaneously condition and primedentin and enamel. The bonding mechanism is based onhybridization, with the difference that only submicron hybridlayers are formed and resin tag formation is less pronounced(9,10).

With the self etching primers, an acidic monomer in theprimer demineralises the smear layer and underlying dentinresulting in mild surface etching. While with 5th generation

Descriptive :

Group Number Mean

Excite (1) 12 14.70

Prompt L pop (2) 12 13.55

Clearfil Liner Bond 2V (3) 12 19.53

Comparisons in Mpa

Group (I) Group (J) Mean difference p (I-J)

Excite Clearfil LinerBond 2v -4.8333 .000 VHS

Excite Prompt L Pop 1.1481 .137 N S

Clearfil LinerBond 2v Prompt L Pop 5.9815 .000 VHS

Shamina Bawa


83

system that is simple, easily retrievable and techniqueinsensitive.

ConclusionThe purpose of this study was to evaluate the dentin

shear bond strength of three step total etch adhesive systemsin comparison with two self etching primer adhesive syatems.It may be concluded that the shear bond strength of dentin isdependent on material (adhesive system), substrate depthand adhesive/depth interaction and not just on the adhesivesystem used.

In the present study, the highest mean shear strengthvalue was produced by Clearfil Liner Bond 2V (19.53MPa),followed by Excite (14.7 MPa) and Prompt-L-Pop([13.55Mpa).

Major developments in adhesive materials, especiallydentin bonding systems are seen in the past years.Conventional three step total etch adhesives remain theadhesive of choice for routine clinical use because of theirleast technique sensitivity. Self etching primers do not requirethe rinsing phase which truly saves time and is less prone tomanipulation errors. However, the clinician should choosethe adhesive system that is simple, easily retrievable andtechnique insensitive.

References1. Mj Tyas, MF Burrow.Adhesive restorative materials:A review, Australian Dent

Jour 2004:49 (3) : 112-121.2. Sano H, Kanemura N, Burrow MF, et al. Effect of operator variability on dentin

Adhesion: students vs. dentists. Dent Mater J 1998;17:51-58.3. Kijsamanmith K, Timpawat S, etal. Microtensile bond strengths of bonding

agents to pulpal floor dentin. Int Endod J 2002;35:833-839.4. Barnes et al. The adhesive revolution of restorative dentistry. Honk kong medical

journal 1996;Vol 2 No.2.5. Guilherme C.P. and Luiz N.B.Dental adhesion: Present state of the art and

future perspectives. Quintessence Int 2002:33,213-2246. Wang Y, Spencer P. Hybridization efficiency of the adhesive / dentin interface

with wet bonding. J Dent Res 2003; 30:99-105.7. Van meerbeek et al.Adhesives and Cements. Oper dentistry supplement

6.2001,119-144.8. Shono Y. and Ogawa T. Regional measurement of resin dentin bonding As a

Array. Jour dent Reaserch 1999: 78(2), 699-7059. Jorge Perdigao, Ronald F. and Bruno T.: New trends in dentin /enamel

adhesion.Am J Dent 2000:13:250-30010. John Kanca III. Bonding to dentin. Clues to mechanism of Adhesion. Am J Dent

1998; vol 11:154-159.11. Akagawa Hirotoshi, Toru Nikaido,Tsunehiko Takada: Shear bond strengths to

coronal and pulp chamber floor dentin. Am J Dent. 2000;15(6) 383-388

total etch adhesives, priming and bonding procedures arecombined in one step. Phosphoric acid is used to remove thesmear layer and a moist surface is required to maintain anon collapsed mineralized collagen network of etched dentinwhich is mediated by water. Usually one bottle adhesivecontain ethanol or acetone as solvent which assess theirapplication to the moist dentin surface.

Akagawa H. et al(11) studying the shear bond strength tocoronal and pulp chamber floor dentin observed that thescanning electron microscope observation of the dentinsurfaces treated with phosphoric acid were quite differentfrom those with self etching primers. For, phosphoric acidetching, the smear layer overlaying the intertubular dentinwas completely removed and the dentinal tubules wereopened widely, where as for Clearfil Liner Bond 2V, the smearlayer was removed. The smear plugs partially remainedbecause of the weaker demineralization. Such morphologicaldifferences are believed to be due to the different acidity ofeach solution, with phosphoric acid having a pH of less than1.0 where as Clearfil Liner Bond 2V primer has a higher pH of2.0.

In this present investigation, we could demonstratehighest shear bond strength value of 19.5 MPa with ClearfilLiner Bond 2V. Comparatively lower bond strengths valueswere obtained with Prompt L Pop, a self etching primer andExcite, a 5th generation total etch adhesive. The results ofthe present study indicate that Clearfil Liner Bond 2V wouldbe a good choice since it produced fairly good bond strengthvalues and is less technique sensitive. However bondstrengths alone are not sufficient to evaluate the ability ofresins on bonding systems.

Clinically, self etching systems not only simplify thebonding process by eliminating steps, but also eliminatesome of the technique sensitivity of total etch systems. Finallythe risk of incomplete resin infiltration is eliminated bysimultaneous infiltration of the exposed collagen fibrilscaffold along with same depth of demineralization.

With the introduction of self etching primers and neweradhesive resin system and bonding techniques it is hopedthat in future these can be adapted for use in many clinicalcondition. However , the clinician should choose the adhesive

Comparative evaluation of the shear bond strength of conventional

total etch adhesive with simplified bonding systems


84

However after 10 yrs when patient visited the clinic thetooth was symptomatic with spontaneous pain, tendernessto percussion and palpation but with no signs of fistula, andno mobility.

Since the patient was symptomatic endodontic treatmentwas decided .Following local infiltrative anesthetics, rubberdam was placed. Endodontic access was attained throughthe crown. The diamond abrasive point was used for theceramic portion and transmetal bur used for the metalportion.

Once the canal orifice was detected the orifice wasenlarged with gates glidden drill upto (size2-3). Bleedingfrom the canal suggested partially vital pulp.

Both the coronal and the apical fragments were includedto the endodontic preparation. The root canal was preparedto size 25 using standardized instrumentation techniquesince there was obliteration of root canal present. Irrigationwas performed with 2% sodium hypochlorite. Final irrigationhas been carried out with saline.The root canal was filledwith gutta percha and sealer (Adseal,Meta Biomed Co.Ltd)using lateral condensation technique. Three months afterthe treatment clinical and radiographic examination of thetraumatized tooth revealed favorable prognosis. The toothwas free of symptoms and presented physiologic mobility.

Discussion

Diagnosis

Diagnosis of root fracture is accomplished by clinicaland radiographic examination. Clinical examinationincludes the evaluation of mobility, the presence or absenceof tenderness and pain to palpation of the soft tissues,percussion of the teeth and pulp testing.(5)

Radiographic demonstration of a horizontal root fracturemay require multiple radiographs. A recent study by

Post healing complication of horizontal fracture : A case report* Swathi Amin, ** Prathap M.S., *** Harish Shetty

Introduction

Radicular fracture in permanent teeth is uncommoninjuries among dental traumas, comprising 5-7% of cases(1, 2)

The leading cause of root fractures injuries was falls(46%) and mostly involved one tooth (71%). Maxillary centralincisors were the most often affected teeth (95%). The mostcommon type of root fracture was in the middle third of theroot (57%) followed by apical part (34%)(3). Coronal fractureare least common with occurrence rate of <15%(4).

Horizontal root fracture in the permanent dentition hasbeen reported to comprise from 0.2% to 7% of all traumaticinjuries to teeth(4).

Root fractures are also more likely to occur in fullyerupted permanent teeth with closed apices in which thecompletely formed root is solidly supported in bone andperiodontium.

A traumatic injury in a deciduous tooth or one with anincompletely formed root with less periodontal support willmost likely result in a luxation or avulsion injury ratherthan a root fracture. Although horizontal root fractures arenot common, the sequelae can be complex because of thecombined damage to pulp, dentin, cementum, bone andperiodontium.(4)

Case report

A 27 yr old female patient came to the dental clinic withpain on maxillary left central incisor which had a porceleinfused to metal crown (PFM). Patient gave a history of roadaccident 10 yr back and went for a PFM crown due to Ellisclass II fracture without undergoing proper radiographicexamination.

Soft and hard tissue examination showed no signs ofscarring or any other evidence indicate precision trauma.Thetooth was asymptomatic for 10 yrs.

Radiographic examination revealed horizontal rootfracture in between apical and middle third. Patient hadreported to her dentist 20 days after the accident, but fracturewas undetected.

* Asst.Professor** Associate Professor*** Professor and HOD, Dept. of Conservative Dentistry and Endodontics, Yenepoya

Dental College, Yenepoya University,Deralakatte, Mangalore - 575 018

Radicular fracture in permanent teeth are uncommon injuries among dental trauma, comprising 0.5 to 7% ofthe cases. This case report describes a post healing complication of an untreated horizontal root fracture ofmaxillary left central incisor which had healed in ten years. The fracture was diagnosed ten years later whenpatient visited the clinic with pain in relation to the same tooth.

Abstract

Case reports


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Anderson6 demonstrates the need for both a periapical andocclusal radiograph, using the bisecting angle technique inorder to ensure the visualization of the root fracture. It wasreported that an occlusal exposure was ideal for disclosingfractures in the apical third of the root and periapicalexposures were better for visualizing coronally located rootfractures. Anderson concluded that the direction of fractureline changes from an oblique angle in the apical and middlethird of the root to a more horizontal fracture line in thecervical third. Thus the occlusal radiograph would result ina central beam direction that would more likely follow asteep oblique line found in an apical or middle third fracture,whereas the less steeply angled periapical radiograph wouldbetter visualize the more horizontally angled coronalfractures. Failure to expose both radiographs may result ina misdiagnosis.(4)

Occasionally even with the use of multiple radiographs,demonstration of the root fracture is not always detectableimmediately after injury (4).

Subsequent radiographs may later demonstrate thefracture through ensuing root separation due to hemorrhageor ingrowth of granulation tissue in the fracture.

In the present case, endodontic treatment was carriedout although electric pulp test was not carried out due to thePFM crown. The patient was symptomatic and tooth wastender on percussion.

Radically both fragments were treated because there wasthe risk that the apical fragment was already infected orcould be infected during treatment.

Research, however indicates that healing is more frequentin horizontal root fracture teeth when endodontic treatmentof only the coronal fragment is carried out compared withwhen endodontic treatment of both of the fragment is carriedout.(7)

In the mentioned study, failure was associated often withprotrusion of gutta percha into the space between thefragments, unlike the case presented here; gross protrusionof endodontic filling materials did not occur and only

tolerable amount of sealer protruded and that did notcompromise the study.(7)

Fracture healingHealing sequelae following root fracture have been

described by Anderson and Hjorting Hansen. They describedfour sequlae:

1. healing with calcified tissue2. healing with inerproximal connective tissue3. healing with interproximal bone and connective tissue4. interproximal inflammatory tissue without healing.(5)

Healing with calcified tissue

The hard tissue union is characterized radiographicallyby the close approximation of the coronal and apicalsegments, with the fracture line being somewhat indistinct(4). Because the apical segment of pulp almost alwaysremains vital following a root fracture, the type of healing isdependent primarily on the amount of damage to and theresponse of the coronal pulp4.

When the pulpal damage is minor and the pulp remainsvital the odontoblast dominate the healing of the fracturewith some assistance from cells of the cementum. This resultsin deposition of hard tissue and a subsequent hard tissueunion of the segment (4).

Inter proximal connective tissueConnective tissue healing demonstrates that the segments

are separated either by a distinct radiolucent fracture lineor by lamina dura and bone. It is not always possible toplace a fracture precisely into one of these two categoriesbecause the healing may consists of both hard and soft tissuecomponents. Clinically, both methods of healing usuallyresults in asymptomatic non mobile teeth. Occasionally,some minor mobility may be associated with connectivetissue healing (4).

When the pulp damage is greater, the odontoblastscannot respond and the predominant cells for healing come

Fig 3: ObturationFig 2: Working LengthFig 1: Diagnostic Radiograph showinghorizontal root fracture

Post healing complication of horizontal fracture : A case report


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from the periodontal ligament, the damaged pulp heals byrevascularization and the fracture heals primarily byfibroblast activity.(4)

Interproximal inflammatory tissue withouthealing:

In 1967, Anderson and Hjorting Hansen introduced whatbecame known as the four “classic categories” of root fracturehealing. This is actually nonhealing and should not beincluded in the classification of healing.(4)

If the pulpal damage is severe, the pulp will becomenecrotic and prevent revascularization. This results in non-healing and nonunion of the fractured segments.(4)

Fractured roots that radiographically revealed less spacebetween the fragments indicates healing more frequentlywith hard tissue or combination of bone and connectivetissue. In the present case, healing would have been hardtissue or combination of bone and connective tissue tenyears back. But after the root canal treatment, since the pulpwas extirpated, the healing was thought to be throughdeposition of cementum by the PDL originated tissue. Similarcase has been reported by Sibel Kocak et al(7)

In a study done by Miomir Cvek et al (8) on the Survivalor healing of fracture, out of 425 teeth new injury occurredin 47 teeth out of which 21 healed spontaneously and 4 afterendodontic treatment. Something similar is seen in this casewhere the patient is thought to have a new injury (hit by thehead of her child) which would have been the reason forpost healing complication.

Pulp canal obliteration:Partial or complete obliteration of the pulp canal is a

common finding after root fracture. Found in 69 to 73% ofthe teeth. Both obliteration types progress at the same rateand are normally well advanced after 9 to 12 months andapproached full density 1 to 2 yrs later.(1)

Two types of hard tissue formation were observed in thepulp, one is progressive apposition of hard tissue on thedentinal walls which slowly obliterate the pulpal lumen.This type is seen in coronal fragments, apical fragments orboth. Second type where hard tissue formation was localizedand clearly related to the fracture site while rest of the pulpallumen remained of normal size and appearance.(8) In thiscase there was partial pulp canal obliteration. Similarfindings have been reported by Jantier.A.Herweijer et al. (9)

References1. Andreasen JO. Traumatic injuries of the teeth. 2nd ed. Philadelphia WB Saunders

1981.2. Poi WR, Manfrin TM, Holland R, Sonoda CK. Repair characteristics of horizontal

root fracture: a case report. Dent Traumatol 2002;18:98-102.3. K.Caliskan MK, Pehlivan Y. Prognosis of root fractured permanent incisors.

Endod Dent Traumatol 1996;12: 129-136.4. Eric J Hovland. Horizontal root fractures, Treatment and Repair . DCNA 1992;

36:509-525.5. Oztan MD, Sonat B. Repair of untreated horizontal root fractures. Two case

reports. Dent Traumatol 2001;17: 240-43.6. Andreasen FM, Andreasen JO: Resorption and mineralization process following

root fractures of permanent incisors. Endodontics and Dental Traumatology4:202,1988

7. Sibel Kocak, Serban Cinar, M Murat Kocak, Guven Kayaoglu. Intra radicularsplinting with endodontic instrument of horizontal root fracture – case report .Dent Traumatology 2008;24:578

8. Miomir Cvek, Georgios Isilingaridis, Jens Ove Andreasen. Survival of 534 incisorsafter intra alveolar root fracture in patient aged 7 -17 years. Dent Traumatology2008,24:379-387.

9. Jantin A Herweijer, Mahmoud Torabinejad, Laif K Bakland. Healing of HorizontalRoot Fractures. Journal of Endodontics. 1992;18: 118-122.

Swathi Amin


87

Revascularization of an immature non-vital permanent tooth- A case report

* Eva.C.Das

The endodontic management of immature permanent teeth creates a lot of challenges because of the incom-plete root formation, open apex and thin friable root canal walls. The condition is even more complicated incase of loss of vitality, as there will not be any vital pulp tissues available for vital pulp therapies. There maybe destruction of the Hertwig's epithelial root sheath, which acts as a guide in root development. Conven-tional apexification procedures often render the roots prone to fractures, and MTA apexification also neededartificial strengthening of roots with composite restoration. A paradigm shift in the management of non-vitalimmature permanent teeth was introduced which was an unconventional, biological regenerative procedurecalled revascularization or revitalization. It revitalizes a necrosed tooth which gives the ideal outcome ofcontinued root formation, both in length and in width, by hard tissue deposition. Here is a case report ofsuccessful revascularization of non-vital immature permanent teeth using a triple antibiotic paste for pulpspace disinfection and MTA.

Abstract

* Senior Lecturer, Dept. of Conservative Dentistry and Endodontics, SreeMookambika Institute of Dental Sciences, Kulasekaram,K K District,Tamil Nadu.

IntroductionIn our clinical practice, it is not rare that we come across

the need for treating an immature permanent teeth, with anopen apex. Depending on the degree of vitality of the tooth,different conventional treatment options are available .These include vital pulp capping, pulpotomy andapexogenesis, and treatment of non vital teeth withapexification. In case of apexification, neither theconventional procedure using calcium hydroxide nor thesimplified procedure using Mineral Trioxide Aggregate (MTA)provides the advantage of continued development of the rootboth in thickness and in length. Moreover the existing rootstructure is weakened by the long term use of calciumhydroxide dressing. Both creates an apical barrier againstwhich routine root canal filling materials can be condensedand the root needs to be strengthened using endodontic posts.Such a procedure can render root canal retreatment difficultif needed [1,2].

One of the rationale of endodontic therapy is thethorough disinfection of the root canal space, after which,establishment of an environment conducive of hard tissueformation can hypothetically, result in the deposition ofdentin, cementum, or similar hard tissues within the rootcanal space, thereby strengthening the tooth from within. Atthis context, comes a new procedure called root canalrevascularization or revitalization, which when successfullydone, can result in revitalizing the tooth as well as enhancingthe development of root both in length and in width. Adetailed case report is presented, which describes therevitalization of a non-vital immature permanent maxillarycentral incisor.

Case ReportA fifteen year old girl was referred for the endodontic

treatment of maxillary left central incisor. She presented

with an Ellis class II fracture of maxillary left central incisor.The patient gave a history of trauma, 5-6 years back afterwhich, occasional pain was reported, which subsided afterabscess formation and drainage. The patient has reportedafter a recent episode of pain. The tooth showed mildtenderness on palpation and slight yellowish discoloration.An evidence of sinus tract was seen in relation to 21(fig: 1).Periodontal probing showed an intact epithelial attachment,normal probing pocket depths, and normal range of mobility.

The radiograph of the affected tooth showed thin rootswith blunderbuss apex, with adjacent teeth showing normaldevelopment for the age, and apparently normalperiradicular tissues (fig:2). A periradicular radiolucencyapproximately of 3mm diameter was present in relation to21. Both electric and thermal pulp testing gave no responsesuggestive of a non-vital tooth.

Treatment planConsidering the age of the patient and assessing the

restorability of the affected tooth, a treatment option ofrevascularization was made. Informed written consent wasobtained before commencement of the treatment.

After local anesthesia and rubber dam isolation, the toothwas accessed. At the first visit the root canal space wasaccessed and disinfected with copious irrigation of 5.2%sodium hypochloride. Minimal instrumentation was doneso as to prevent the fracture of already weakened root canalwalls. The pulp space was dried and a triple antibiotic pastemade up of equal quantities of tetracycline, amoxicillin andmetronidazole mixed with 2% chlorhexidine was placed. Theaccess preparation was closed with Glass Ionomer Cement.The patient was recalled after 1 week.


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Eva C. Das

At the next appointment, the root canal space wasaccessed and the tripple antibiotic paste was washed offwith normal saline, the root canal space was irrigated with10ml of 5.2% sodium hypochloride solution for 10 minutes.The pulp space was dried with sterile paper points. A sterile25 gauge injection needle of 1.5" was inserted into the rootcanal space until it reached the periradicular area to inducebleeding into the root canal space. Once bleeding wasinduced till the cemento-enamel junction, grey MTA wasmixed and placed inside the coronal pulp space and theaccess cavity was restored with light cure compositerestoration. A radiograph was taken to confirm the extendof the MTA. The patient was recalled after 6 months. Thetooth was asymptomatic clinically, and showed radiographicevidence of reduction in the size of periradicularradiolucency and continued root formation. (Fig:3)

Discussion:Experimental studies have been done in animals and the

success in re-vascularization in animal lead to the use ofthis procedure in immediately replanted avulsed immaturepermanent teeth [3]. An immature or an open apex tooth hasthe potential to establish revascularization when there isminimum of 1.1 mm apical opening [4] .Complete pulp canalrevascularization has been shown to occur at a rate of 18%among immature teeth [5]. Furthermore, when extra oral timeis limited to no more than 5 minutes, revascularization amongteeth with open apices in dogs has been found to be as highas 91% with topical minocycline and 73% with topical

doxycycline. Antibiotic treatment appears to reducecontamination of root surface or pulp space thereby creatinga biologic environment that aids revascularization [6,7,8].

A mature tooth or teeth with a closed apex has little or nochance of pulp revascularization so far. Studies on traumaticinjuries to immature teeth show promise forrevascularization of root canal system if the size of the apicalforamen is 1.1mm or more [3,4]. In the present case, traumato the incompletely developed central incisor has lead to thenecrosis of the pulp, resulting in the cessation of rootdevelopment. Apexification procedures can form an apicalbarrier against which root canal obturation can be done,but it does not allow for the natural strengthening of the rootthrough hard tissue formation along the length and width ofthe tooth. That is the reason why revascularization wasattempted in this case.

One of the key factor for the success of revascularizationis disinfection of the root canal system to create anenvironment conducive to hard tissue formation [9, 10]. Thiswas achieved by the use of Sodium hypochloride as irrigantand calcium hydroxide and chlorhexidine as well as tripleantibiotic paste as intracanal medicaments for pulp spacedisinfection (11). The triple antibiotic paste used in this casewas a combination of a broad spectrum (tetracycline) , agram positive spectrum (amoxicillin) and an anaerobicspectrum (metronidazole) antibiotics. Tetracyclines arebacteriostatic, which is advantageous in the absence ofantigenic byproducts such as endotoxins are not releasedinto the pulp space. They inhibit mammalian collagenasesand clastic cells, which result in anti resorptive activity.Tetracycline have smear layer removing capacity and exhibitsubstantivity for upto 12 weeks (12). Metronidazole has abroad spectrum of action against protozoa, anaerobic cocci,obligate anaerobes and gram negative and gram positivebacilli [11] .Chlorhexidine was used as a carrier medium aswell as an adjuvant to the antimicrobial agent as it too hasa bactericidal action, substantivity and it prevents thebreakdown of collagen of dentine [11]. Minimalinstrumentation was done so as not to weaken the alreadythinned out root dentin.

Once, bacteria free canal is achieved tissue will not growinto empty space but needs an environment which promotesthe regeneration of new tissue. At this context, comes theconcept of the regenerative triad, that includes the cellsscaffolds, and the mediators, which when acting in a mutuallycomplimentary manner, can initiate revitalization of the pulpspace in a time bound manner [12, 13]. In the present case,this was provided by the blood clot by inducing bleedingfrom the periradicular tissue into the pulp space, which actedas an active scaffold that contains the cells and mediatorsfor the growth of new tissue [14, 15].

The cells that are capable of regeneration of the hardtissues inside the pulp are the post natal stem cells, whichare capable of self renewal, proliferation and differentiationinto multiple specialized cell lineages within the dental pulp,apical papilla and periodontal ligament. These cells includethe post natal dental pulp stem cells (DPSCs), stem cells ofexfoliated deciduous teeth (SHED), stem cells of apical papilla(SCAP) and periodontal ligament stem cells (PDLSC). Theability of these cells to pulp-dentine and cementumperiodontal ligament complexes in vivo suggests the

Fig 1. Pre operative photograph showing Ellis Class IIfracture of 21.

Fig 3. 6 months follow-upradiograph showing continuedroot formation and reduction

in radiolucency.

Fig 2. Pre operative radiographshowing open apex and

periradicular radiolucency inrelation to 21.


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possibility of regeneration of hard tissue on providing properscaffolds and mediators [16].

The mediators include secreted proteins that bind to cellreceptors and modulate cellular activity. Fibroblast growthfactor (FGF), transforming growth factor beta (TGFβ),superfamily including bone morphogenic proteins (BMPs),platelet derived growth factor (PDGF) and Insulin like growthfactor (IGF) have overlapping and functions in stem cellcontrol and regulation of proliferation [16].

Appropriate biodegradable scaffolds which are capableof containing or being seeded with growth factors and bioactive signaling molecules, supporting cell organization, andgrowth of a vascular supply are to be provided for theregeneration of pulp or periodontal tissues. Natural scaffoldslike collagen offer good biocompatibility and bioactivity.Artificial synthetic copolymers like D, L-lactide and glycolidecan be fabricated with impregnated with growth factors ormediators [17].

In the case of immature permanent teeth, the idealsituation is the presence of surviving pulp, Hertwig’sepithelial root sheath(HERS) and vital cells of apical papillaafter root canal disinfection. Continued root formation toits maturity and an increased thickness of root dentine maythen be anticipated in such case. In the present case, traumamust have resulted in the necrosis of pulp and disruption ofHertwig’s epithelial root sheath, and apical papilla, but byproviding a sterile environment, fluid tight seal, and a bloodclot that contains a scaffold of fibrin network containinggrowth factors and cells with regenerative potential, othertissues are capable of filling the pulp space.

The mechanisms of revascularization are hypothesizedto be multifactorial. In case of a few vital cells remaining atthe apical foramen; they may proliferate into the newlyformed matrix from the blood clot and differentiate intoodontoblasts, which can lay down a tubular dentin. Themultipotent dental pulp stem cells, stem cells of theperiodontal ligament, stem cells of apical papilla or thebone marrow stem cells from instrumentation beyond theconfines of the root can enter the root canal space throughthe open apex, and can deposit tertiary or atubular dentine,cementum or even osteoid tissue, depending on thepredominant cell type [18, 19]. The blood clot itself, being agood source of growth factors can induce hard tissueformation.

Once disinfection and blood clot formation is complete,the next step is to protect the blood clot with a fluid tightseal. MTA was placed over the blood clot at the level of CEJand a moist cotton pellet was placed over the MTA. After theMTA set, the moist cotton pellet was removed and acid etchcomposite was placed over this. Shah et al has reported theuse of glass ionomer cement to provide a fluid tight seal,without any osteoinducive materials inside the pulp spacewith promising results, which is a cost effective alternativeto MTA, but further studies have to be made [18]. Jung et al inhis case studies has reported continued root formation withand without clot formation [19].

Follow up evaluation was done at 6 months. Follow upevaluation included radiographs. Inspection for sinus tractor abscess formation, palpation to rule out mobility andtenderness. In the present case, the radiographs revealedevidence of continued apical development, radiopacities

within canal space associated with partial canalobliteration, suggestive of hard tissue deposition, andnarrowing of canal space suggestive of continued rootdevelopment. Since the nature of the hard tissue formed canbe confirmed only after histological examination, it wasdecided that as long as the tooth is asymptomatic, withradiographic evidence of hard tissue formation, no furthertreatment was needed.

ConclusionDepending on the data available, it should be concluded

that any immature teeth with a necrotic or partially necroticor infected pulp which needs a pulpectomy and orapexification procedures should be given a chance to berevitalized with the new procedure called revascularization.Should it fail, there are other options of apexification (eitherwith calcium hydroxide or MTA), but revascularization ofthe tooth should be used as the first line of treatment.Whether the new tissue formed is truly pulp or pulp-like is oflittle consequence, as long as there is continued developmentof the root canal walls and apex, which strengthens the toothagainst fracture naturally. If subsequent necrosis or infectionoccurs, routine root canal therapy can be undertaken andprognosis will be better than with an open apex [20].

References1) Andreasen JO, Farik B, Munksgaard EC. Long term calcium hydroxide as a root canal

dressing may increase risk of root fractures. Dental Traumatology 2002; 18:134 - 7.2) Kalebzadeh N, Dalton B, Trope M. Strengthening immature teeth during and after

apexification. J. Endod. 1998; 24:256-9.3) Yanipset K, Trope M. Pulp revascularization of replanted immature dog teeth after

different treatment methods. Endodontic Dental Traumatology 2000; 16:211-7.4) Kling M, Cvek M, Mejare I. Rational predictability of pulp revascularization in

therapeutically reimplanted permanent incisors. Endodontic Dental Traumatology1986; 2:83-9.

5) Ritter AL, Ritter AV, Murreh V, Sigurdssen A, Trope M. Pulp revascularization ofreplanted immature dog teeth after treatment with minocycline and doxycyclineassessed by Laser Doppler Flowmetry, radiography and histology. Endodontic DentalTraumatology 2004; 20:75-84.

6) Cvek .M, Cleaton-Jones .P, Austin J, Lownie J, Kling M, Fatti P. Effect of topicalapplication of doxycycline on pulp revascularization and periodontal healing inreimplanted monkey incisors. Endod. Dental Traumatology 1991; 6:170-6.

7) Cvek .M, , Cleaton - Jones P, Austin .J, Lownie J, Kling .M, Fatti .P. Pulp revascularizationin reimplanted. Immature Monkey incisors – Predictability of antibiotic systemicprophylaxis. Endodontic Dental Traumatology 1990; 6:157-69.

8) Banchs F, Trope M . Revascularization of Immature Permanent Teeth with ApicalPeriodontitis -New treatment protocol J Endod. 30; 4:196-200.

9) Kakehashi .S, Stanley HR, Fitzgerald RJ. The effects of surgical exposure of dentalpulps in germ free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol.1965;20:340-9.

10) Sandquist G, Bacteriologic studies of necrotic pulps. Umea , Sweden.11) Mohammadi Z, Abbot P V. On the local applications of antibiotics and antibiotic-

based agents in endodontics and dental Traumatology. International endodonticJournal,42,555-567,2009.

12) P.E. Murray, F. Garcia Godoy K.M. Hargreaves Regenerative endodontics: A review ofcurrent status and a call for action. J Endod. 2007; 4:377-87.

13) Iwaya SI, Ikawa M, Kubota M. Revascularization of an immature permanent toothwith apical periodontitis and sinus tract. Dent. Traumatol 2001;17:185-7.

14) Marx RE. Platelet Rich Plasma: Evidence to support its use J. Oral Maxillofac Surg.2004;62:489-96.

15) Freymiller E.G, Aghatoo TL. Platelet Rich Plasma: Ready or not J. Oral Maxillofac Surg2004;62:484-88

16) Friedlander L T,Cullinan M P,Love R M.Dental stem cells and their potential role inapexogenesis and apexification. International Endodontic Journal,42,955-962,2009.

17) Huang G T J.Apexification: the beginning of its end.International EndodonticJournal,42,855-866,2009.

18) Shah N, Logani A,Bhaskar U, Aggarwal V. Efficacy of revascularization to induceapexification/apexogenesis in infected non vital immature teeth: a pilot clinical study.JEndod.34,919-25,2008.

19) Jung I Y, Lee S J, Hargreaves K M. Biologically based treatment of immature permanentteeth with pulpal necrosis: a case series. J Endod. 34,876-87,2008.

20) Blayne T, Trope M. Pulp revascularization of a necrotic infected immature permanenttooth: case report and review of literature. . J. Endod. 2005; 546-50.

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IntroductionThe oral cavity is a battlefield of activities of

demineralization and remineralization. Dental caries is ahighly prevalent diet-related disease commonly affecting thetooth structure. The prevalence of the disease has decreasedsince the introduction of modern dentistry. A goal of moderndentistry is to manage non-cavitated caries lesions non-invasively through remineralization in an attempt to preventdisease progression and improve aesthetics, strength, andfunction of the teeth (1).

RemineraliaztionThe mineral in human enamel & dentin is not pure

hydroxyapatite, but rather a mixture of compounds includinga number of carbonated apatite’s, which together occupy upto 98 weight per cent and upto 96 volume per cent, whenconstitutional water is included. Remineralization is definedas “the process whereby calcium and phosphate ions aresupplied from an external source to the tooth to promote iondeposition into crystal voids in demineralized enamel, toproduce net mineral gain”(2). The reason for the developmentof the dental caries is now recognized as an “imbalance inthe demineralization & remineralization cycle”, which is anatural phenomenon under the biofilm that is attached tothe tooth surface. The ratio of components required forremineralisation is 10 calcium ions to 6 Phosphate ions toeither 2 fluoride or 1 hydroxyl ions or 1carbonate ion, aratio of 5:3:1. An ideal remineralizing agent should diffuseinto the subsurface or deliver calcium and phosphate into

the subsurface, should not deliver an excess of calcium,should not favour calculus formation, should work at anacidic pH and boost the remineralizing properties of saliva(3).

DemineralizationDemineralization is the dissolution of calcium salts by

lactic acid followed by decomposition of the gelatinous bodyof the organic matrix. “Dental caries is an infectiousmicrobial disease that begins with the demineralization ofinorganic portion of tooth followed by destruction of organicportions, leading to cavity formation”. Dental Caries is amultifactorial disease consisting of four main parameterscontributing for its initiation- the host factor (tooth),Microorganisms (mainly Mutans streptococci), diet (sucroserich) & time. Demineralization and remineralization is acontinuous process. When the bacteria act upon thecarbohydrates, certain acids (like lactic acid) are produced.These acids diffuse through calcified dental tissues resultingin dissolution of the apatite crystallites. Bacteria producelactic acid until the local ph reaches about 4.5-5 at whichtime they cease to form any more lactic acid.

Dentin is an anisotropic composite possessing superiormechanical properties. They contain approximately 70wt%

Dental caries is highly a prevalent disease that take places due to imbalance between demineralization andremineralization. With the introduction of modern dentistry our goal is to prevent the disease progressionnoninvasively. A new paradigm shift occurred in the thought about remineralization of dentin, that for com-plete remineralization both the inter and intrafibrillar remineralization should occur. Conventionalremineralization strategy depends on epitaxial growth over existing apatite crystallites. If there are no or fewcrystallites, there will be no remineralization. Guided tissue remineralization uses biomimetic analogs ofdentin matrix proteins to introduce sequestered amorphous calcium phosphate nano precursors into theinternal water compartments of collagen fibrils. Attachment of templating analogs of matrix phosphoproteinsto the collagen fibrils further guided the nucleation and growth of apatite crystallites within the fibril. Such astrategy is independent of apatite seed crystallites. Guided tissue remineralization is an effective method forcomplete remineralization of dentin and such formed dentin will have better hierarchical assembly of apa-tites within collagen matrix.

Abstract

Guided tissue remineralization: A novel method for toothremineralization

*Sapna C M, ** Baby James, ***Rakesh, **** Sreedevi N B

* Reader** Professor and HOD*** Senior Lecturer**** P.G. Student,

Department of Conservative Dentistry and Endodontics,Amrita School of Dentistry, Amrita Institute of MedicalScience, Cochin.

Review


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mineral phase, 20 wt% organic phase and 10wt%water.Odontoblast, which are specialized dentin forming cells,synthesize the major constituents of dentin matrix namelytype 1 collagen and non collagenous proteins. This type 1collagen constitutes 90 wt% of dentin organic phase andremaining 10% is composed of non collagenous proteins(NCP’s). Acid produced by bacteria that is the lactic acidtends to preferentially dissolve some but not all interfibrilarcrystallites creating zones of partially demineralized dentin.They also activate the endogenous collagenolytic enzymesknown as the matrix metalloproteinase’s (MMP’s) that breakdown the demineralized collagen matrix(4).

Dentine remineralizationThe idea of dentin remineralization is certainly not new;

the dental literature abounds with studies of various fillingmaterials that are capable of remineralizing demineralizeddentin. Conventional remineralization of artificial cariousdentin often involves the use of calcium and phosphate ioncontaining solutions in presence of various concentrationsof fluoride. It is well established that conventionalremineralization does not occur by spontaneous nucleation

of mineral on the organic matrix but rather by growth ofresidual apatite seed crystallites (5). This may be regardedas the top down approach and may be regarded as classicalcrystallization theory via ion by ion addition to pre-existingseed crystallites. Non classical crystallization via bottomup approach is the particle mediated mesoscopictransformation possibly followed by fusion to an iso-oriented crystal and finally to a single crystal 6. In case ofclassical crystallization the mineral content of the lesionsurface layer influences the characteristics of subsequentremineralization, including the location and density ofmineral deposition. Although fluoride enhances mineraluptake, it causes hyper mineralization of the lesion surfaceand prevents effective remineralization of the deeper partsof the carious lesion. Thus, slightly elevated fluoride levelsare considered less effective in preventing lesion progressionin dentin than in enamel(5).

The mineral phase in mineralized dentin is classified asintrafibrillar and extrafibrillar. Intrafibrillar apatites aredeposited within the gap zones of collagen fibril and extendalong the microfibrillar spaces within the fibril. Extrafibrillarapatites are deposited within the interstitial spaces

(Reference 6) (Reference 11)

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separating the collagen fibrils(7). Intrafibrillarmineralization contributes significantly to the mechanicalproperties of dentin. The end-point for assessing success orfailure of remineralization by mineral density alone has beenchallenged as heterogeneous precipitation of extrafibrillarapatite contributes minimally to the mechanical propertiesof remineralized dentin(9).

Classical crystallization versus non classical crys-tallization

Classical crystallization starts from primary buildingblocks like atoms, ions or molecules, by forming clusters,which may grow or disintegrate again, depending on thecounter play of surface and surface lattice energies.Eventually, some clusters reach the size of a so called Criticalcrystal nucleus. At this stage the free energy of systembecomes negative up on further particle growth, because thegain in lattice energy over compensate the loss in surfaceenergy.

Hence for complete remineralization the better optionwill be non classical crystallization ,here the events arealways particle mediated and involve mesoscopictransformation process. A mesocrystal is colloidal crystalthat are composed of individual nano crystals that arealigned in common crystallographic fashion, exhibitingscattering properties similar to single crystal. The mainpathway include the arrangement of primary nano particlesinto an iso-oriented crystal via oriented attachment whichcan form a single crystal upon fusion of the nano particles.If the nano particles are coated by some organic componentsthey can form a mesocrystal via mesoscale assembly,possible followed by fusion to an iso-oriented crystal andfinally to single crystal.

Non classical crystallization offers some peculiaradvantages with respect to crystal morphologenesis suchas near independence of solubility products and indifferenceto ph and osmotic pressure(6).

Guided tissue remineralizationGuide tissue remineralization (GTR) represents a novel

strategy in collagen biomineralization. This strategy utilizesnanotechnology and biomimetic principles to achieveintrafibrillar and extrafibrillar remineralization of a collagenmatrix in the absence of apatite seed crystallites(11). In thisstrategy, two polyanionic analogs are involved to mimic thesequestration and templating functions of matrix proteinsin biomineralization. This particle-mediated bottom-upmineralization strategy is different from conventionalremineralization techniques currently employed in dentistryin two aspects (11,12). Firstly, it is a biomimetic processthat recapitulates the progressive dehydration mechanismof natural biomineralization by replacing free and looselybound water within a collagen matrix by apatite crystallitesvia the use of polyanion- stabilized amorphous calciumphosphate nanoprecursors(13). Secondly, this particle-based assembly approach proceeds in the absence of apatiteseed crystallites in a collagen matrix. Whereas epitaxialgrowth over existing seed crystallites is a thermodynamicallymore favourable process.

Mineralization in the absence of seed crystallites requiresalternative kinetically-driven protein/ polymer-modulatedpathways for lowering the activation energy barrier forcrystal nucleation via sequential steps of phasetransformations, as depicted by non-classical crystallizationtheory(14).

Materials used for GTR1) Portland cement2) Poly vinyl phosphonic acid3) Poly (aspartic) acid and poly acrylic acidPortland cement - a bioactive cement, when interacted

with phosphate containing fluid produces calcium-deficientapatites via an amorphous calcium phosphate phase(15).

Both Poly(aspartic) acid and polyacrylic acid arecarboxylic acid-containing polyelectrolytes which have been

(Reference 5)

Sapna C M


93

employed as analogues of acidic non-collagenous proteinssuch as dentin matrix protein 1 (DMP1) for stabilising andcontrolling the dimensions of amorphous phases in calciumcarbonate and calcium phosphate precipitation systems(16).

Polyvinylphosphonic acid (PVPA) is a polyelectrolyte thathas been employed as a biomimetic analogue forphosphoproteins such as DMP1 and dentinephosphoprotein. It has been shown that natural dentinephosphoprotein and its analogue protein phosvitin did notprecipitate apatites when they were available freely inmetastable calcium phosphate solutions, but they only doso when immobilised on the collagen substrate(17).

How remineralization occurs with guided tissueremineralization.

In GTR remineralization occur mainly by three steps1) Induction step2) Recruitment step3) Meso scopic assemblyIn the first nanoprecursor induction step, addition of

polyacrylic acid to the Portland cement–PCF systemproduces metastable amorphous calcium phosphatenanoprecursors which are small enough to penetrate ademineralised collagen matrix (figure -a). These nanosphereshave the potential to be transformed into apatitenanocrystals which are probably also stabilised bypolyacrylic acid anions.

This step occurs irrespective of whether PVPA is present.However, in the absence of PVPA, there is very limitedattraction of these nanoprecursors into the demineralisedcollagen matrix. Recombinant DMP1 molecules were thoughtto perform the dual functions of calcium phosphate bindingand collagen binding by having specific sites on the moleculefor performing these functions. As a biomimetic molecule,polyacrylic acid probably simulates the calcium phosphatebinding sites of DMP1 but not its collagen binding sites.

In the second controlled nanoprecursor recruitment step,inclusion of PVPA probably results in the immobilisation ofthese polyelectrolyte anions along the collagen microfibrillsas well as on the surface of the collagen fibrils, in a mannerthat is analogous to the interaction between dentinephosphoprotein and collagen. Thus, PVPA is additionally

required to simulate the collagen-binding function of DMP1in guiding nanoprecursor recruitment to the collagen matrix.As apatite nanocrystals are deposited within the collagenfibrils, they appear corrugated, revealing the periodicity ofthe fibrils. Apatite nanocrystals are directed by theimmobilised PVPA anions to orientate along the microfibrills(intrafibrillar remineralisation) and the surface of thecollagen fibrils (interfibrillar remineralisation). Thenanocrystals that are initially deposited are much smallerthan the apatite platelets found in natural intact humandentine. These nanocrystals represent the primary buildingunits in dentine remineralisation. Thus it is necessary to useboth of the biomimetic analogues to guide the deposition ofintrafibrilar apatites within collagen fibrils

In the third hypothetical mesoscopic assembly step, thePVPA-anchored nanocrystals probably guide other apatitenanocrystals to orientate by self-assembly, resulting in largepolymer-stabilised mesocrystals. These mesocrystalseventually transform via a particle-mediated ‘‘non-classicalcrystallisation mechanism’’ into larger apatite plateletswithin and along the surface of the collagen fibrils.(11)

DiscussionApatite deposition within collagen fibrils appears to be

regulated by two important steps. The first step issequestration of calcium phosphates into nanoscopiccompartments which is referred to as polymer-stabilisedamorphous mineral phases(18). Recent studies have shownthat the first mineral formed in mineralised collagenoustissues is a liquid phase of amorphous calcium phosphate(ACP) that can be drawn into the nanoscopic gaps of collagenfibrils by capillary action (19,20).

The subsequent step relies on the role of matrixphosphoproteins in providing templates to control thedimension and hierarchy of apatite deposition withincollagen fibril. Incorporation of a biomimetic analogue withsequestering function into a mineralisation medium helpsstabilise ACP precursors in their required nanoscaledimensions. The use of phosphoprotein biomimeticanalogues further serve as templates that guide the self-assembly of the stabilised nanoprecursors along specificsites on and within collagen fibrils. Studies havedemonstrated that polyanionic macromolecules can initiateamorphous to crystalline phase transition in a controlledmanner. These phenomena are thought to proceed via thenonclassical crystallisation pathways of bottom-upnanoparticle assembly and mesocrystalline transformation(12).

Conclusion

To date, no conventional remineralization studies havedemonstrated complete remineralization of artificial cariouslesions from lesion surface to the base. From the recentstudies it is evident that both intrafibrilar and extra fibrillarremineralization is essential for complete remineralization

Sapna C MGuided tissue remineralization: A novel method for tooth remineralization

(Reference 11)


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of dentin(5). Despite reports on dentine remineralisationusing phosphoprotein analogues, casein phosphopeptide-amorphous calcium phosphate and nanometric bioactiveglass particles, there has been no report on definitiveintrafibrillar remineralisation of demineralised dentinecollagen matrices using biomimetic approaches(11). Fromthis basic concept of complete remineralization muchprogress has been made in understanding aboutbiomineralization process. Collagen fibrils in dentin are thebasic building blocks of mineralized hard tissue. Thecollagen matrix does not initiate mineralization of its ownand is regulated through interaction between collagen matrixand non collagenous proteins(12). Acid non collagenousproteins control crystal nucleation and the dimension, orderand hierarchy of apatite deposition within mineralized hardtissues but the therapeutic use of native or recombinantextracellular matrix proteins for in-situ biomineralizationis economically prohibitive. Thus research scientist haveelected using polyanionic macromolecules that mimic thefunctional domains of theses naturally occurring proteins(21).

The new biomimetic mineralization strategy (guide tissueremineralization) developed is based on binding of twobiomimetic analogues to collagen fibrils so that the dopedcollagen can guide the scale and distribution ofappatites(11). Biomimetic analogues- poly acrylic acid cancreate meta stable ACP nano precursors in presence ofcalcium and phosphate ions at a high Ph(22). Polyvinylphosphonic acid mimics the negative charge ofphosphoprotien DMP1, bone sialoprotein and phosphoryn.The bound analogues present multiple negatively chargedsites for calcium binding at both intrafibrilar andinterfibrilar locations(23). In addition to biomimeticanalogues, set Portland cement is used for sustained releasehydroxyl and calcium ions when it interacts with phosphatecontaining fluid to produce carbonated apatites via ACPphases. A high Ph of mineralization medium is required forautotransformation of ACP into more stable apatite phasewithout the participation of an octacalcium phosphate phasethat is formed at Ph values less than 9.25(15). Recent studyused this strategy and found that the interaction betweenthe collagen and biomimetic analogues lead to the formationof mineralized collagen fibrils that approximate thedimensions and order of those found in natural mineralizedtissues within 24hrs (12). Another study evaluated theefficacy of guided tissue remineralization in theremineralization of acid etched dentine that is incompletelyinfiltrated by dentine adhesives as well as partiallydemineralized caries affected dentine and foundintrafibrillar and interfibrillar remineralisation of 5 mmthick layer of demineralised dentine (11). Thus theunderstanding that biomolecule control the

biomineralization give a new insight into the controlledsynthesis of mineralized tissues and the formed structurescould successfully reproduce the structural hierarchy ofnaturally occurring mineralized tissues.

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16) He G, Gajjeraman S, Schultz D, Cookson D, Qin C, Butler WT, et al.Spatially andtemporally controlled biomineralization is facilitated by interaction betweenself-assembled dentin matrix protein 1 and calcium phosphate nuclei in solution.Biochemistry 2005;44:16140–8.

17) Olszta MJ, Odom DJ, Douglas EP, Gower LB. A new paradigm for biomineralformation: mineralization via an amorphous liquid-phase precursor. ConnectTissue Res 2003;44(Suppl. 1):326–34.

18) Gower B. Biomimetic model systems for investigating the amorphous precursorpathway and its role in biomineralization. Chem Rev 2008;108: 4551-627.

19) Boskey AL. Amorphous calcium phosphate: the contention of bone. J Dent Res1997;76:1433-6

20) Olszta MJ, Odom DJ, Douglas EP, Gower LB. A new paradigm for biomineralformation: mineralization via an amorphous liquid-phase precursor. ConnectTissue Res 2003;44:326-34.

21) Zhang W, Liao SS, Cui FZ. Hierarchical self-assembly of nano-fibrils in mineralizedcollagen. Chem Mater 2003;15:3221-6

22) Liou SC, Chen SY, Liu DM. Manipulation of nanoneedle and nanosphere apatite/poly(acrylic acid) nanocomposites. J Biomed Mater Res B Appl Biomater2005;73:117-22.

23) He G, Gajjeraman S, Schultz D, Cookson D, Qin C, Butler WT, et al. Spatially andtemporally controlled biomineralization is facilitated by interaction betweenself-assembled dentin matrix protein 1 and calcium phosphate nuclei in solution.Biochemistry 2005;44:16140-8.

Sapna C M


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FORMAT AND STRUCTURE

Format

Language: The language of publication is English. Authorsare strongly encouraged to analyze their final draft withboth software (e.g., spelling and grammar programs) andcolleagues who have expertise in English grammar. TheEditor reserves the right to edit all manuscripts or to rejectthose manuscripts that lack clarity or precision, or haveunacceptable grammar.

Presentation: Authors should pay special attention to thepresentation of their research findings or clinical reportsso that they may be communicated clearly. Technical termsshould be avoided as much as possible and clearlyexplained where its use is unavoidable. Abbreviationsshould also be kept to a minimum, particularly those thatare not standard. The background and hypothesesunderlying the study, as well as its main conclusions,should be clearly explained. Titles and abstractsespecially should be written in language that will bereadily intelligible to any scientists.

Abbreviations: JCAESOK adheres to the conventions outlinedin Units, Symbols and Abbreviations: A Guide for Medicaland Scientific Editors and Authors. When non-standardterms appearing 3 or more times in the manuscript are tobe abbreviated, they should be written out completely inthe text when first used with the abbreviation inparenthesis.

Structure

All manuscripts submitted to JCAESOK should include TitlePage, Abstract, Main Text, References andAcknowledgements, Tables, F igures and F igure Legendsas appropriate

A. Title Page: The title page should bear: (i) Title, whichshould be concise as well as descriptive;. It is best not touse abbreviations in the title since this may lead toimprecise coding by electronic citation programs (ii)Authors’ full names, highest academic degrees andaffiliations (iii) Name, full postal address, telephone,fax number and e-mail address of author responsible forcorrespondence (iv) All sources of support, includingpharmaceutical and industry support that requireacknowledgment.

B. Abstract for Original Scientific Articles should be no morethan 250 words giving details of what was done using thefollowing structure:• Aim: Give a clear statement of the main aim of the studyand the main hypothesis tested, if any.• Methodology: Describe the methods adopted including,as appropriate, the design of the study, the setting, entryrequirements for subjects, use of materials, outcomemeasures and statistical tests.• Results: Give the main results of the study, including theoutcome of any statistical analysis.• Conclusions: State the primary conclusions of the studyand their implications.

This section often is written last by many authors sincethey can draw on the rest of the manuscript. Write the

abstract in past tense since the study has been completed.Three to ten keywords should be listed below the abstract.

C. Main Text of Original Scientific Article should includeIntroduction, Materials and Methods, Results, Discussion,Conclusion and References.Introduction: The introduction should briefly review thepertinent literature in order to identify the gap inknowledge that the study is intended to address. Thepurpose of the study, the tested hypothesis and its scopeshould be described. Material and Methods: The objective of the methods sectionis to permit other investigators to study and replicateyour experiments. The three components to this portionare the experimental design, the methods employed, andthe statistical tests used to analyze the results. The vastmajority of manuscripts should cite prior studies usingsimilar methods and succinctly describe the particularaspects used in the present study. If the study utilized acommercial product, the manuscript should state that theyeither followed manufacturer ’s protocol or specify anychanges made to the protocol. Studies on humans shouldconform to the Helsinki Declaration of 1975 and statethat the institutional IRB approved the protocol and thatinformed consent was obtained. Studies involvinganimals should state that the institutional animal careand use committee approved the protocol. The statisticalanalysis section should describe which tests were usedto analyze which dependent measures; p-values shouldbe specified. The soft ware used to analyse the data hasto be mentioned. Additional details may includerandomization scheme, stratification (if any), poweranalysis, drop-outs from clinical trials, etc. Results: Only experimental results are appropriate in thissection (i.e., neither methods nor conclusions should bein this section). Include only those data that are criticalfor the study. Do not include all available data withoutjustification; any repetitive findings will be rejected frompublication. All F igs./Charts/Tables should be describedin their order of numbering with a brief description of themajor findings.Discussion: may usefully start with a brief summary of themajor findings, but repetition of parts of the abstract orof the results section should be avoided. The Discussionsection should progress with a review of the methodologybefore discussing the results in light of previous work inthe field. The Discussion should end with a briefconclusion and a comment on the potential clinicalrelevance of the findings. Statements and interpretationof the data should be appropriately supported by originalreferences.Conclusion: should contain a summary of the findings.References: The reference style follows Index Medicus. Eg-Mc Cracken MS, Haywood VB. Demineralization effects of10 percent carbamide peroxide. J Dent 1996; 24:395-8. Thereferences are numbered as it appears in the article andplaced in parentheses at the end of a sentence or at theend of a clause that requires a literature citation. Do notuse superscript for references. Original reports are limitedto 35 references. There are no limits in the number ofreferences for review articles.

Guidelines to Authors


96

ARTICLE SUBMISSION TO JCAESOK

Manuscripts submitted for publication must be submittedsolely to the JCAESOK and not published elsewhere.

Manuscript Submission: The prepared article and the coverletter should be sent as separate files [email protected]

Preparation of Manuscript: Manuscripts that do not adhereto the instructions will be returned to the correspondingauthor for technical revision before undergoing peerreview.

Title Page: The title page must be submitted as a separatefile. Include on the title page: (a) Complete manuscripttitle; (b) Authors’ full names, highest academic degreesand affiliations; (c) Name and address for correspondence,including fax number, telephone number and e-mai laddress; address for reprints if different from that ofcorresponding author; and (d) All sources of support,including pharmaceutical and industry support that requireacknowledgment.

Abstract: The abstract and text must be submitted as aseparate file. Limit the abstract to 250 words. It must befactual and comprehensive. Limit the use of abbreviationsand acronyms, and avoid general statements (e.g., “thesignificance of the results is discussed”). List three tofive keywords or phrases.

Text: Organize the manuscript into six main headings:Introduction, Materials and Methods, Results, Discussion,Conclusion and References. Define abbreviations at firstmention in text and in each table and figure. If a brandname is cited, supply the manufacturer ’s name andaddress (city and state/country). References follow thestyle of Index Medicus. They must be keyed to the text andnumbered consecutively in the order of appearance.References should be placed inside parentheses at theend of a sentence or a clause, and not placed assuperscripts.

Figures: Art should be created/scanned and saved andsubmitted as either a TIFF (tagged image file format), anEPS (Encapsulated PostScript) file or a PPT (PowerPoint)file. Line art must have a resolution of at least 1200 dpi(dots per inch); all scanned images including electronicphotographs/radiographs, CT scans, etc., must have aresolution of at least 300 dpi. If fonts are used in theartwork, they must be converted to paths or outlines, orthey must be embedded in the files. Publication of colorillustrations is possible and, at the editor’s discretion.

Tables: Tables should be double-spaced with a single boldruling beneath the column titles. Units of measurementsmust be included in the column title.

Figures: All figures should be planned to fit within either1 column width (8.0 cm), 1.5 column widths (13.0 cm) or 2column widths (17.0 cm), and must be suitable forphotocopy reproduction from the printed version of themanuscript. Lettering on figures should be in a clear.Figures should be on a white background, and shouldavoid excessive boxing, and unnecessary colour, shading.

The line spacing should be wide enough to remain clear

on reduction to the minimum acceptable printed size.

Figure legends: Figure legends should begin with a brieftitle for the whole figure and continue with a shortdescription of each panel and the symbols used; theyshould not contain any details of methods. Hard copiesof all figures and tables are required when the manuscriptis ready for publication. These will be requested by theEditor when required. Each Figure copy should be markedon the reverse with the figure number and thecorresponding author’s name.

Cover Letter: A cover letter, containing signatures of allauthors and the following information, must be scannedand submitted along with the article word document.a. The manuscript title, name and address (including e-mail) of one author designated as the correspondingauthor. This author will be responsible for editing proofsand ordering reprints when applicable. b. The followingparagraph: “In consideration of the editors of the JCAESOKtaking action in reviewing and editing this submission,the author(s) undersigned hereby transfer, assign orotherwise convey all copyright ownership to the CAESOKin the event that such work is published in that Journal.”c. If the purpose of a paper is to evaluate a commercialproduct, then a separate statement must be included withthe submission, which asserts that the product was usedexactly according to manufacturer ’s instructions. If thiswas not the case, a precise description of any variant usemust be prominently stated in the abstract, methods and,if appropriate, in the title. d. All authors must also signthe following statement, which must accompany themanuscript: “I affirm that I have no financial affiliation(e.g., employment, direct payment, stock holdings,retainers, consultantships, patent licensing arrangementsor honoraria), or involvement with any commercialorganization with direct financial interest in the subjector materials discussed in this manuscript, nor have anysuch arrangements existed in the past three years. Anyother potential conflict of interest is disclosed.” Anyauthor who cannot sign this statement must append aparagraph to the manuscript that fully discloses anyfinancial or other interest that poses a conflict. Thisparagraph should follow the “Discussion” section. e. Ifhuman subjects are used, include the following statement:“ The informed consent of all human subjects whoparticipated in the experimental investigation reportedor described in this manuscript was obtained after thenature of the procedure and possible discomforts andrisks had been fully explained.” If animals are used, astatement on protocol approval by the institutional animalcare and use committee must be included.

Disclaimer: “The statements, opinions and advertisementsin the JCAESOK are solely those of the individual authors,contributors, editors or advertisers, as indicated. Thosestatements, opinions and advertisements do not effectany endorsement by CAESOK or its agents, authors,contributors, editors or advertisers, or the publisher.Unless otherwise specified, the CAESOK and the publisherdisclaim any and all responsibility or liability for suchmaterial.”

issn 2249-4812 vol 1 no.2 oct 2011 for web...jcaesok the journal of conservative and endodontic...

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