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BALKAN JOURNAL

OF STOMATOLOGYOfficial publication of the BALKAN STOMATOLOGICAL SOCIETY

ISSN 1107 - 1141

Volume 16 No 2 July 2012

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BALKAN JOURNAL OF STOMATOLOGY ISSN 1107 - 1141

STOMATO

LOGIC

AL

SOCIETY

ALBANIA

Ruzhdie QAFMOLLA - Editor Address:Emil KUVARATI Dental University ClinicBesnik GAVAZI Tirana, Albania

BOSNIA AND HERZEGOVINA

Maida GANIBEGOVI - Editor Address:Naida HADIABDI Faculty of DentistryMihael STANOJEVI Bolnika 4a

71000 Sarajevo, BIH

BULGARIA

Nikolai POPOV - Editor Address:Nikola ATANASSOV Faculty of DentistryNikolai SHARKOV G. Sofiiski str. 1

1431 Sofia, BulgariaFYROM

Julijana GJORGOVA - Editor Address:Ana STAVREVSKA Faculty of DentistryLjuben GUGUEVSKI Vodnjanska 17, Skopje

Republika MakedonijaGREECE

Anastasios MARKOPOULOS - Editor Address:

Haralambos PETRIDIS Aristotle UniversityLambros ZOULOUMIS Dental School

Thessaloniki, Greece

ROMANIA

Alexandru-Andrei ILIESCU - Editor Address:

Victor NAMIGEAN Faculty of DentistryCinel MALITA Calea Plevnei 19, sect. 1

70754 Bucuresti, Romania

SERBIA

Dejan MARKOVI - Editor Address:Slavoljub IVKOVI Faculty of DentistryZoran STAJI Dr Subotia 8

11000 Beograd, Serbia

TURK EY

Ender KAZAZOGLU - Editor Address:Pinar KURSOGLU Yeditepe UniversityArzu CIVELEK Faculty of Dentistry

Bagdat Cad. No 238Gztepe 81006Istanbul, Turkey

CYPRUS

George PANTELAS - Editor Address:Huseyn BIAK Gen. Hospital NicosiaAikaterine KOSTEA No 10 Pallados St.

Nicosia, Cyprus

Editorial board

Editor-in-Chief Ljubomir TODOROVI, DDS, MSc, PhDFaculty of DentistryUniversity of BelgradeDr Subotia 811000 BelgradeSerbia

Council:

President: Prof. H. Bostanci

Past President: Prof. P. Koidis

President Elect: Prof. N. Sharkov

Vice President: Prof. D. Stamenkovi

Secretary General: Prof. A.L. Pissiotis

Treasurer: Prof. S. Dalampiras

Editor-in-Chief: Prof. Lj.Todorovi

Members: R. QafmollaP. KongoM. GanibegoviS. KostadinoviA. Filchev

D. Stancheva ZaburkovaM. Carev

A. MinovskaT. Lambrianidis

S. Dalambiras

A. AdiM. DjurikoviN. FornaA. BucurM. CareviM. BarjaktareviE. Kazazoglu

M. AkkayaG. PantelasS. Solyali

BALKAN STOMATOLOGICAL SOCIETY

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The whole issue is available on-line at the web address of the BaSS (www.e-bass.org)

International Editorial (Advisory) Board

Christoph HMMERLE - Switzerland George SANDOR - CanadaBarrie KENNEY - USA Ario SANTINI - Great BritainPredrag Charles LEKIC - Canada Riita SUURONEN - FinlandKysti OIKARINEN - Finland Michael WEINLAENDER - Austria

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BALKAN JOURNAL

OF STOMATOLOGYOfficial publication of the BALKAN STOMATOLOGICAL SOCIETY

ISSN 1107 - 1141

Volume 16 No 2 July 2012

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BALKAN JOURNAL OF STOMATOLOGY ISSN 1107 - 1141STOMA

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SOCIETY

LR P. Papadopoulos Langerhans Cell Histioctosis: 69Its Oral and Maxillofacial Dimension

OP M. Pandilova Effects of Local Application of Ascorbic Acid and 74

A. Ugrinska Glutathione b Iontophoresis on Gingival Inflammation

S. Georgieva

M. Popovska L. Kanurkova K. Smilevska

OP L. Gavriliuc Salivar Glutathione-Dependent Enzmes in Patients with 79

E. Stepco Dental Fluorosis Treated b Complex Antioxidant Therap

I. LupanN. Sevcenco

I. Spinei

OP A. Dimkov Release of Antimicrobial Agents from 84E. Gjorgievska Glass Ionomer Cements

A. Fildishevski

OP E. Boteva Efficac of Working Length Detection and 90

D. Yovchev Irrigation during Preparation of Curved Root Canals

OP T.T. Akbay Salivar Thromboplastic Activit Ma Indicate 94

M. Guvercin Wound Healing after Tooth ExtractionO. Gonul

A. Yarat

S. Akyuz

R. Pisiriciler

K. Gker

OP D. Veleski Biophsical Principles of the AcrLock Attachments Use in 98B. Pejkovska Contemporar Prosthetic DentistrM. Antanasova

CR K. Tolidis Intraoral Ceramic Restoration Repair Techniques: Report of 3 Cases 103

P. Gerasimou

C. Boutsiouki

CR B.Vanlolu A Multidiscipline Approach to Improve Aesthetics in a Patient with 109

Y. zkan High Lip Line: A Clinical Report

Y. Kulak-zkan

Contents

VOLUME 16 NUMBER 2 j ULy 2012 PAGES 65-128

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CR L. Mavriqi The Use of Advanced Technolog to Avoid Inur of the 112E. Baca Inferior Alveolar Nerve during Implant SurgerA. Vjeshta

CR S. Karamanis Bon Lid Approach in Dentoalveolar Surger: Report of 2 Cases 116

D. Tsoukalas

A. Traskas

N. Parissis

CR S. Dalampiras Technique of Frenectom of Labial Fraenum with 122C. Boutsiouki Combined Osteotom at Intermaxillar Suture

CR B. Evrosimovska Fracture of the Maxillar Tuberosit: A Case Report 125B. VelickovskiZ. Menceva

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SUMMARYThis paper reviews clinical, histological and pathological characteristics

of Langerhans Cell Histiocytosis (LCH) in relation to its oral and maxillofacial

interest. For purposes of better examination, LCH is differentiated into 3

syndromes: Hand-Sculler-Cristian (HSC) disease, Letterer-Siwe (LS) disease

and Eosinophilic Granuloma (EG). Abnormal proliferation of histiocytic cells

with Birbeck granules is found in all 3 of them.The cause of LCH is related to abnormal proliferation of Langerhans

Cells (LCs) which are mononuclear phagocyte system cells concentratedin oral gingival sulcular and junctional epithelium. LCH appears to attackpatients of any age, especially young children. Its predilection sites arethe maxilla and the mandible. In LS disease, ulceration of oral mucosa,premature loss of teeth, suppuration or haemorrhage, all are commonsymptoms. Moreover, in HSC soreness, generalized stomatitis and bonedestruction are found. EG preserves all the above characteristics andunderlines a delayed healing after extraction and pathologic fractures.

The diagnosis is served by radiographic images, which show solitaryintraosseous lesions, defined periphery and multiplicity of alveolar bonelesions, bone sclerosis or root resorption. Early diagnosis improves theprognosis and effectiveness of the treatment. The weapons against LCH are:surgery, corticosteroids and alkylating agents.

Kewords: Langerhans Cell Histiocytosis; Hand-Sculler-Cristian Disease; Letterer-SiweDisease; Eosinophilic Granuloma; Alveolar Bone; Bone Destruction

Petros Papadopoulos

Private Practice

LI TERATURE REVIEW (LR)

Balk j Stom, 2012; 16:69-73


Langerhans Cell Histiocytosis:Its Oral and Maxillofacial Dimension

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Introduction

The term Histiocytosis X was introduced by Lichten-stein in 1953 for a group of diseases that produce 3syndromes with similar clinical and histopathologicalfeatures1: Hand-Sculler-Cristian (HSC) disease, Letterer-Siwe (LS) syndrome and Eosinophilic Granuloma (EG).Since all 3 diseases have an abnormal proliferation ofhistiocytic cells with characteristic Birbeck granules, adifferentiation between them has been abandoned and theterm Langerhans Cell Histiocytosis (LCH) is commonlyused nowadays2.

LCH develops in childhood as well as in adulthood3.It can involve many organ systems but primarily affects

bone, skin, lymph nodes, lung, liver and spleen, endocrineglands and nervous system4.

Its dental interest derives from its special histo-pathological characteristics referring to the oral and

maxillofacial region, as well as its relationship to certaindental pathologies. This comprises the reason of a reviewof all the aforementioned syndromes, which can be provenuseful either for general dentists or for specialized ones.

Role of Langerhans Cells in Oral

Pathology

Langerhans Cells (LCs) are bone marrow derivedcells that belong to the mononuclear phagocyte system

and (or) dendritic system5

. In gingiva they are present in3 areas: (1) the oral gingival epithelium; (2) the sulcular

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70 Petros Papadopoulos Balk J Stom, Vol 16, 2012

epithelium; (3) the junctional epithelium. They are locatedin the suprabasal and spinous layers of the oral gingival andsulcular epithelium. In the junctional epithelium, 2 typesof Langerhans cells can be observed. The first type witha spherical morphology possesses a few short dendrites

and is weakly stained for Castlemans Disease (CD) 1aThymocyte (T) 6 antigen. The other type exhibits dendritesof moderate length and number with varied fluorescenceintensity against CD1a T6 antigen6. These heterogeneitiescould represent different stages of a dynamic processleading to an accumulation in number of LCs7.

Much experimental evidence has shown that thereare differences both in LC numbers and in surfaceantigen expression between healthy and diseasedgingiva. Consequently, LC could represent key cells inpathogenesis and development of periodontal disease8.Moreover, the relationship between an increased number

of LCs and plaque accumulation was demonstrated in menduring experimental gingivitis. More LCs were found inthe inflamed gingiva in comparison to healthy gingivafrom the same patients. Many studies also suggested anincrease of the LCs in moderate gingival inflammation,but a decreased number of LCs in periodontitis comparedwith the controls. The numerical diminution of LCsin the passage from gingivitis to periodontitis revealsan important role of these cells in the pathogenesis ofgingival disease.

In contrast to gingivitis and periodontitis, asatisfactory explanation of the proliferation of LCs in thelesions of LCH is still missing, although its appearance isdefinitely related to this proliferation9.

Histology (Microscopy)

For purposes of a better examination of the histologyof the disease, its differentiation into the 3 syndromeswe mentioned in the introduction could be critical.Consequently:1. Hand-Schuller-Cristian disease (chronic disseminated)

comprises of multiple lesions in the bones and soft

tissues initially, with some visceral lesions developinglater8. Except of the skin lesions, which are typicalnodules in flexures, a crusted scalp rash mimickingseborrheic dermatitis may also be seen. Oral lesionsinclude gingivitis, ulcerations and destructivegranulomas involving the mandible and the maxilla.Gingival tissue shows a dense infiltrate (histiocytic)throughout most of the connective tissue. The nucleiof the histiocytes are, for the most part, regular withsome vacuolisation. There is a significant number ofeosinophils scattered throughout much of the infiltrate.

2. Letterer-Siwe disease (acute disseminated): It

demonstrates an excessive proliferation of histiocytesthat accumulates in tissues. It can also include

nodular foci. Oral manifestations such as gingivitisand ulcers as well as oral bleeding and eventual lossof teeth are present10.

3. Eosinophilic granuloma of bone (chronic focal): Itcan be solitary or multifocal11.

LCs are associated with eosinophils and often othertypes of granulocytes in variable number. LCs havepale, vesiculated, weakly eosinophilic cytoplasm andnuclei that appear folded or lobulated. Mitotic activityis typically absent. Eosinophils may be scattered amongthe LCs or histiocytes. By electron microscopy, Birbeckgranules may be seen in LCs. They appear as lamellarplates with a central striated line. They occasionally havea terminal vesicular dilatation giving them a racquetshape.

Incidence and Dental Predominant

Sites

LCH is considered a childhood disease, but thediagnosis is often made in adults as a likely evolution ofthe juvenile form12. It seems to be more frequent in malesthan in females, with a reported ratio ranging from 1:1to 4:113. In adults, there is a much greater variation witha slight predominance of female patients12. It is veryinteresting to notice that in a review of 1120 patients,Hartman reported oral involvement in 10% of the cases.

a. LS disease: There is no special information about theincidence of the LS disease. It particularly affectsinfants or young children, under the age of 2 years,predilection sites being the alveolar parts of themaxilla and the mandible;

b. HSC disease occurs mainly in children or adults. Themaxilla and the mandible may be the first structuresto show signs of this condition14. It commonly affectsolder children, between ages of 5 and 10, but may beseen in any group;

c. EG constitutes about 50% to 60% of all HistiocytosisX cases15. It is a disease with an incidence of 1 new

case/350000-2 million per year16, 17.Approximately 75% of all patients are below 20

years of age2. Predominant locations are the flat bones,with frequent involvement of the mandible in patientsless than 20 years old. Individuals over the age of 50 areuncommonly affected.

Oral Manifestations

In case of the LCH, the first manifestations and

symptoms occur in the mouth5

and identification isnecessary for the diagnosis of the disease. The leading

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symptom of the LCH within mandibular and maxillarybones is pain, which sometimes is misdiagnosed as amarginal infection2. There is also loosening of teeth,as common presenting complains of the patients, aswell as necrotising and ulcerating defects of the mucosa

and jaw swelling5. The ulcerations are accompaniedby granulomatous exophytic tissue in the areas of theattached gingiva in the maxilla, extending to the palateanteriorly and posteriorly18.

In clinical cases without bone involvement, palatal,lingual and vestibular bilateral ulcerations were recordedin molar maxillary and mandibular regions. Submucosalnodules were also recorded in the superior and inferiorfrontal gums19. Except from pain,, a burning sensationand spontaneous and mechanically induced bleedingduring oral hygiene procedures were noticed. The upperand lower third molar regions were more inflamed and

painful19

.More specifically referring to each entity of thedisease separately, we can look into the followingsigns and symptoms of oral involvement of the LCH:(1) The LS syndrome -ulcerations of oral mucosa,diffuse destruction of bone, premature loss of teeth,haemorrhage, foul breath, suppuration; (2) The HSCdisease - generalised stomatitis, soreness, haemorrhagefrom the gums, ulceration and necrosis of the oral mucosa,progressive bone destruction of the alveolar process,loosening and premature loss of teeth, facial asymmetry;(3) The EO - periodontitis localised in an otherwisehealthy dentition, loss of alveolar bone with the area ofdestruction replaced by soft tissue, delayed healing afterextraction teeth, premature loss of teeth, foul breath,solitary soft tissue involvement may affect the tongue andmay also be confused with traumatic granuloma20.

In the EO, pathologic fractures may occur especiallyin the long bones15. It is also useful to underline the factthat in the HSC disease, typical lesions of the diseaseinvolve the cranial bones, the eyes (exophthalmia) andthe pituitary gland (diabetes insipidus), whereas in theLS disease, the syndrome is characterised by lymphnode, spleen and liver involvement, with a severe clinicalcourse12.

Diagnosis - Radiographic Image

An effort to find out the diagnosis of LCH wouldbe surely incomplete and insecure without taking intoconsideration the radiographic image of the disease. LCHpresents as localised punched out radiolucencies with nocalcification and no sign of sclerosis or reaction at theborders. There may be severe alveolar bone resorptionproducing an appearance of teeth floating in space.

Panoramic radiograph and computerised tomography isused for this purpose21. Moreover, in the EO of the jaws,

the borders of the lesions are mostly well delineated,whereas in the HSC disease the lesions appear as round,oval or irregular areas with sharp margins. In the jaws,these areas look like cysts.

Concentrically, several studies showed at least 7

radiographic characteristics occurred frequently with LCHof the jaws, such as the solitary intraosseous lesions thatare located outside the alveolar process, the multiplicity ofalveolar bone lesions or the well defined periphery. Theperiphery of the lesions of LCH in the jaws is consideredto be well defined but uncorticated.

Another radiographic characteristic is the scoopedout shape. This occurs because bone destruction startsbelow the crest of the alveolar process. Usually, a portionof the superior aspect of the crest of the alveolar bone ismaintained at the mesial and distal margins of the areaof destruction and produces the scooped out appearance.

Bone sclerosis is a common observation in inflammatorylesions of the jaws, and the fact that it appears frequentlyin the alveolar bone lesions might be explained bycommunication of the lesions with the oral cavity, thatresults in a superimposed infection. Periosteal new bone isobserved in intraosseous lesions. The identification of thepresence of this thin layer of bone is highly dependent onthe projections available for study. Finally, root resorptionassociated with lesions of the LCH is always very slight22.

It is necessary to notice the need of biopsies alongwith immunohistochemistry to confirm the diagnosis ofthe LCH and ascertain the nature of cells involved in thelesions detected18. The infiltration cells in LCH are S-100,CD1, CD4 and Human Leukocyte Antigen (HLA)-DRpositive. Electron microscopy will also detect the presenceof Birbeck granules23.

Differential Diagnosis

The puzzle of a successful diagnosis is completed bythe fulfilment of the differential one: clinically, the LCHis difficult to be distinguished from bone metastases,osteomyelitis or even malignant tumours. The final

diagnosis of the LCH can be made only by histology.Morphologically, the cells are characterised by lobulatednuclei, basophilic nuclei and eosinophilic cell plasma.By immunohistochemistry, tumour cells usually expressS-100 and CD-1a. The detection of cytoplasmic inclusionbodies known as Birbeck-Breatuach granules is a typicalcharacteristic of the LCH24. Besides histopathologicdiagnosis, a bone scintigraphy is mandatory to excludeor to detect additional lesions. A common extraosseusmanifestation can be found in the lungs25.

The clinical appearance and course of the presentinglesions usually suggest a differential diagnosis including

other causes of chronic ulceration, such as trauma,necrotizing sialometaplasia, tuberculosis or deep fungal

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infection. Although T-cell lymphoma might manifestas an ulcer, growth is very rapid and progressive, withdestruction of underlying bone. In contrast, in the LCH,the ulcer may maintain the same appearance for months.

Another differential diagnostic possibility is

melanoma, although the presence or not of pigmentationallows clinically to distinguish it from the LCH.

As mentioned above immunohistochemistry is veryuseful to confirm diagnosis. S-100 positivity might provesufficient, in the appropriate light microscopic setting andwith negative immunohistochemical studies for HumanMelanoma Black (HMB)-45, leukocyte common antigen(LCA) or CD3026.

Prognosis

Generally, the prognosis for patients with the variousforms of LCH has improved steadily with the advent ofan early and successful diagnosis and the evolution of amore effective treatment. However, clinical prognosisof patients will become worse with the growing numberof involved organs, with growing number of oraldysfunctions27, with rapid disease progression, withlimited treatment response and decreasing age of the firstdisease manifestation22.

Early onset is associated with bad prognosis.In younger children, before the age of 3, the diseaseprogresses rapidly and is fatal. Late onset is associatedwith milder forms of the disease. Prognosis is excellentin isolated EO of bone, which may heal spontaneously.There is a 90-95% recovery. Prognosis is also good tovery good in multiple EOs restricted to bone15. Moreover,soft tissue involvement is associated with bad prognosis.

In bone and soft tissue involvement, the mortalityrate is 50%. In HSC disease the mortality rate has beenestimated to be 30%. In LS disease the outcome is usuallyfatal. Death usually ensues within 1-3 years from bonemarrow depletion, toxicity, septicaemia haemorrhageand exhaustion. In HSC death usually ensues fromopportunistic infections, intracranial extension and

anaemia. Long survival is an exception in LS disease. Ininfants, it is very acute and rapidly fatal.

Treatment

The treatment of LCH is dependent on the lesionsize and the degree of tissue involvement, and thus differsfrom unifocal or multifocal (monostotic or polyostotic)presentations26. Surgery, in particular for solitary bonelesions, is still the treatment of choice. Following

radiation therapy, patients frequently experience painrelief; however a complete remission is seldom achieved2.

In some large or multifocal lesions, it is necessary tofollow surgical curettage with radiation therapy.

Oral lesions are treated by topical corticosteroids(betamethasone dipropionate 0.05% and sometimes incombination with topical antifungals (miconazole oral gel)

to avoid oral Candida infections. The patient undergoesfrequent oral professional hygiene sessions to minimizemucosal and periodontal damage.

Particularly, in the LS disease with a poor prognosis,therapy consists of steroids used in conjunction witha cytotoxic drug. Alkylating agents provide a moredefinite suppressing action. Vinblastin has proved tobe a medication of value. Moreover, in recent years,drug treatment, especially in cases of multiorganinvolvement, gains more and more importance. Suchdrugs are 2-deoxycoformin, etoposide, vinblastin28-30,mercaptopurine, methotrexate, predisolone, interferon31

and interleukin14

.

Concluding Remarks

As it is well understood, the most importantparameter in the analysis of a disease as a scientific issueis its cure. Generally speaking about cure of the LCH, notonly in jaws but as a multi-system threat, very interestingquestions are born such as: is LCH a malignant or aninflammatory disorder? Should all LCH patients receivetherapy?

Concerning the first question, clinical data isambigual. Clonal expansion of LC, but not lesionalT-cells, was defined by the human androgen receptor(HUMARA) DNA assay as well as T-cell receptoranalysis32. These findings have led many aficionados ofLCH to strongly state that it is a malignant proliferation.Given certain CGH/LOH results from clonal versusnonclonal LCH, the controversy on the malignant natureof the LCH seems far from settled33,34.

About the second question (whether should allLCH patients receive therapy), the studies show distinctconclusions. The simple answer is no when including

single skull lesions in the frontal, parietal or occipitalareas and other skeletal lesions. However, it would be amistake to say that LCH is a slowly progressive diseasein which a wait and see approach should be adopted.This is especially true of pulmonary, jaw and skin diseaseof adults. Smoking cessation may be effective in somepatients with lung disease, but they need to be monitoredcarefully since the insidious progression of cystic changesand fibrosis can rob the patients of vital lung function.

Heroic surgery for jaw disease results indisfigurement and loss of teeth, whereas a 6 monthcourse of viblastine and prednisone can cure the disease

and allow reformation of the bone with no loss ofdentition. Finally, painful and disturbing perineal ulcers

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of LCH in women are best treated with chemotherapy orthalidomide, not radiation.

This whole description reveals the importance of theexistence of such questions as the above. These prove tobe the basis of further evolution of scientific studies about

certain subjects of great medical concern35.

References

1. Lichtenstein L. Histiocytosis: Integration of eosinophilicgranuloma of bone, Letterer-Siwe disease and Hand-Schuller-Cristian disease as related manifestations of asingle nosologic entity. Arch Pathol, 1953; 56:89-102.

2. Eckardt A. Schultze A. Maxillofacial manifestations ofLangerhans cell histiocytosis: A clinical and therapeuticanalysis of 10 patients.J Oral Oncology, 2003; 39:687-694.

3. Howarth DM, Gilchrist GS, Mullan PB, et al. Langerhanscell histiocytosis: Diagnosis, natural history managementand outcome. Cancer, 1999; 85:2278-2290.

4. Mortazavi H, Ehsani A, Namazi MR, et al. Langerhans cellhistiocytosis. Dermatol Online J , 2002; 8(2):18.

5. Schepman KP, Radden BG, Van der Waal I . Langerhans cellhistiocytosis of the jaw bones. Report of 11 cases. AustrDent J , 1998; 43(4):000-000.

6. Lombardi T, Hauser C, Budtz-J orgensen E. Langerhanscells: structure, function and role in oral pathologicalconditions.J Oral Pathol Med, 1993; 22:193-202.

7. Sagredo E, Pino A, Ibanez P. Evaluation de letat gingivalpar la quantification desantigenes T6 (CD1a) et HLA-DR.JBiol Buccale, 1990; 18:163-168.

8. J enney MEM. Langerhans cell histiocytosis: Where do wego from here? Lancet, 1994; 344:1717-1718.

9. Chen HC, Shen WC, Chou DY, et al. Langerhans cellHistiocytosis of the skull complicated with an EpiduralHematoma.AJNR AmJ Neuroradiol, 2002; 23(3):493-495.

10. Strodel BJ . Letterer-Siwe disease: Report of Case. ASDC JDent Child, 1977; 44(4):310-313.

11. Landrito J , Sakurai K, Ohshima K. Use of the ultrasonicsurgical aspirator in the treatment of the solitaryeosinophilic granuloma of the mandible: A case report. JOral Maxillofac Surg, 1990; 48:855.

12. Muzzi L, Pini Prato GP, Ficarrat G. Langerhans cellhistiocytosis diagnosed through periodontal lesions: A Casereport.J Periodontol, 2002; 73:1528-1533.

13. The French Langerhans cell histiocytosis study group.A multicentre retrospective survey of Langerhans cellhistiocytosis: 348 cases observed between 1983 and 1993.Arch Dis Child, 1996; 75:17-20.

14. Thoma KH. Oral Pathology. 4th ed. St Louis: CV Mosby,1954; pp 666-672.

15. Uckan S, Gurol M, Durmus E. Recurrent multifocalLangerhans cell, Eosinophilic Granuloma of the jaws:Report of a case.J Oral Maxillofac Surg, 1996; 54:906-909.

16. Libicher M, Roegen I, Troeger. Localised Langerhans cellHistiocytosis of bone; treatment and follow-up in children.JPediatr Radiol, 1995; 25:134-137.

17. Mc Cowage GB, Frush DP, Kurtzberg J . Successfultreatment of two children with Langerhans cell histiocytosis

with 2-deoxycoformycin.J Pediatr Hematol Oncol, 1996;18:154-158.

18. Bottomley WK, Gabriel SA, Corio RL, et al. HistiocytosisX: Report of an oral soft tissue lesion without bonyinvolvement. Oral Surg Oral Med Oral Pathol, 1987;63:228-231.

19. Manfreddi D, Corradi V, Vescovi P. Langerhans cellhistiocytosis: A clinical case without bone involvement.JPeriodontol, 2005; 76:143-147.

20. Zachariadis N, Anastasea-Vlachou K, Xypolyta A, et al.Uncommon manifestations of histiocytosis X. Int J OralMaxillofac Surg,1987; 16:355-362.

21. Kelly KM, Pritchard J . Monoclonal antibody therapy inLangerhans cell histiocutosis -feasible and reasonable?Br JCancer, 1994; 70(23):54-55.

22. Dagenais M, Pharoah MJ , Sikorski PA. The radiographiccharacteristics of histiocytosis X: A study of 29 cases thatinvolve the jaws.J Oral Surg Oral Pathol, 1992; 74:230-236.

23. OdamRB, J ames WD, Berger TG. Andrews Disease of theskin. 9thed. Philadelphia: WB Saunders Co, 2000; pp 913-917.

24. Chu T, Taffe R. The normal Langerhans cell and the

Langerhans cell histiocytosis cell.J Cancer, 1994; 23:4-9.25. Malpas TS, Norton AJ . Langerhans cell histiocutosis in theadult.J Med Pediatr Oncol,1996; 27:540-546.

26. Milian MA, Bagan JV, J imenez Y. Langerhans cellhistiocytosis restricted to the oral mucosa. Oral Surg OralMed Oral Pathol Oral Radiol Endod, 2001; 91:76-79.

27. Giona F, Caruso R, Testi AM, et al. Langerhans cellhistiocytosis in adults. A clinical and therapeutic analysisof eleven patients from a single institution.J Cancer, 1997;80:1786-1791.

28. Basade MM, Nair CN, Kurkure PA, et al. Etoposide inLangerhans cell histiocytosis in children: a preliminaryexperience. Med Pediatr Oncol,1996; 13:159-162.

29. Gadner H, Heitger A, Grois N, et al. Treatment strategy for

disseminated Langerhans cell histiocytosis. Med PediatrOncol,1994; 23:72-80.

30. Ladisch S, Gadner H, Aric M, et al. LCH-I: a randomizedtrial of etoposide vs. vinblastine in disseminated Langerhanscell histiocytosis. The Histiocyte Society. Med PediatrOncol,1994; 23:107-110.

31. Hirose M, Saito S, Yoshimoto T, et al. Interleukin 2 therapyof Langerhans cell histiocytosis. Acta Paediatr, 1995;84:1204-1206.

32. William CL, Busque L, Griffith BB, et al. Langerhanscell histiocytosis (histiocytosis X) - a clonal proliferativedisease. N Engl J Med, 1994; 331:154-160.

33. Murakami I, Gogusev J, Fournet JC, et al. Detection ofmolecular cytogenetic aberrations in Langerhans cellhistiocytosis of bone. HumPathol,2002; 33:555-560

34. Dacic S, Trusky C, Bakker A, et al. Genotypic analysis ofpulmonary Langerhans cell histiocytosis. Hum Pathol,2003; 34:1345-1349.

35. Mc Clain KL, NatkunamY, Swerdlow SH. Atypical cellulardisorders. Hematology,2004; pp 283-296.

Correspondence and request for offprints to:

Petros Papadopoulos

Th. Sakellaridi 25a

542 48 Thessaloniki, GreeceE-mail: [email protected]

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SUMMARYManagement of gingival inflammation is always a priority in dental

practice, regardless the possible involvement of other periodontal tissues.Besides the use of systemic medical therapy, such as antibiotics, vitamins arealso recommended as a complementary therapy. Nutritional vitamin intakehas been proven effective on gingival inflammation. The aim of this study

was to evaluate the effects of local application of glutathione and ascorbicacid by iontophoresis on gingival tissue inflammation.

60 patients with periodontal disease were divided into 2 groups: thecontrol group was treated by conservative treatment of periodontal diseaseonly, and the study group was treated by ascorbic and glutathione, appliedwith iontophoresis in 10 sessions, besides conservative treatment. Values forgingival inflammation and gingival bleeding on probing were noted.

The obtained results showed significant differences for both gingivalinflammation and gingival bleeding between the examined groups, withsignificant decrease in index values for gingival bleeding after 3 months inthe study group.

Kewords: Gingival Inflammation; Iontophoresis; Ascorbic Acid; Glutathione

Maa Pandilova1, Ana Ugrinska2,Silvana Georgieva1, Mirana Popovska1,L idia Kanurkova1, Katerina Smilevska1

1University Dental Clinical Centre St.Pantelejmon2Faculty of Medicine,Institute of Pathophysiology and

Nuclear Medicine Akademik Isak TadzerSkopje, FYROM

ORIGI NAL PAPER (OP)

Balk j Stom, 2012, 16:74-78


Effects of Local Application of Ascorbic Acid andGlutathione by Iontophoresis on Gingival Inflammation

STOMATO

LOGIC

AL

SOCIETY

Introduction

Managing gingival inflammation is one of the firstchallenges that every peridontologist faces, no matter ifthe process is located only in the gingival tissue, or severeperiodontal destruction had taken place. Furthermore,

control of infection and gingival inflammation isnecessary regardless the following course of periodontaltherapy, conservative or surgical. Keeping inflammationunder control is even more important in the maintenanceperiod.

The methods used for dealing with gingivalinflammation are various, such as: removal of localirritations, training the patients to maintain optimal plaquecontrol, and applying different kinds of medication.Besides the antibiotic therapy, which in certain cases isapplied, for control of inflammation and creating betterconditions for managing microorganism challenge,

vitamins are often used as a supplemental therapy. Theeffects of vitamins on the gingival tissue are confirmed

when taken in natural forms, like fruits and vegetables14.Receiving vitamins as supplements in a chemical way(pills, tablets), according to our previous examinations15did not show measurable results.

Therefore the aim of this study was to verify theeffects, if any, of topical appliance of ascorbic acid and

glutathione delivered by iontophoresis in the gingivaltissue, on gingival inflammation.

Material and Methods

For realization of our goal 60 patients withperiodontal disease, randomly selected, participated in thestudy. The selected patients met the following criteria: None of the patients had attachment lost on probing

greater then 5 mm;

None of the patients had any systematic disease, orwas receiving any drugs;

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Table 2. Average index values for gingival bleeding for thecontrol group

X Sd Se t p

Before therapy

After therapy3 months after therapy

2.76

1.562.20

0.550.72

0.110.09

11.394.26

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76 Maja Pandilova et al. Balk J Stom, Vol 16, 2012

it can be synthesized in the body from the amino acidsL-cysteine, L-glutamic acid, and glycine, and acts directlyas a generic ROS scavenger of the so-called Phase IIreactions17. GSH has multiple functions: it is the majorendogenous antioxidant produced by cells, participating

directly in neutralization of free radicals and reactiveoxygen compounds, as well as maintaining exogenousantioxidants, such as vitamins C and E, in their reducedactive forms20. It is used in metabolic and biochemicalreactions, such as DNA synthesis and repair, proteinsynthesis, prostaglandin synthesis, amino acid transport,and enzyme activation.

Some periodonto-pathogenic bacteria depleteGSH, and this may explain why the amount of thisantioxidant was not elevated in the gingival tissue ofpatients with periodontal disease1,2,10. A similar resultwas obtained in gingival tissue and blood, and lower

levels of GSH were detected in the crevicular gingivalfluid of patients with chronic periodontal disease, whencompared to normal subjects1,2. Systemic depletion ofantioxidants clearly indicates that in chronic periodontaldisease the antioxidant system is affected by a relativelystrong oxidation insult, which can deplete nutritionalantioxidants, such as vitamin E and C in plasma, and alsovitamin E in red cell membrane16.

According to the literature data on the positive effectof this kind of additional therapy with vitamins, and inour previous studies, we confirmed that individuals whichon regular daily base use citrus and citrus like fruits haveless gingival inflammation and gingival bleeding fromthose who never use it, or use it occasionally14. But inour further studies we got to the conclusion that if thevitamins are received chemically (as pills) the significantresults were not achieved15.

In order to be able to get better topic apply ofantioxidants, directly in the gingival tissue, we decided touse permeability of the oral mucosa and enhance the drugappliance by electric current using iontophoresis. Thepermeability barrier in oral mucosa is believed to be theresult of intercellular material derived from the so-calledmembrane coating granules (MCG)6. MCG start formingthrough cell differentiation and at the apical cell surfaces

they fuse with plasma membrane. This barrier is present inthe outermost 200 m of the superficial layer. Permeationstudies have been performed using a number of very largemolecular weight tracers, such as horseradish peroxidaseand lanthanum nitrate3. After being applied to the outersurface of the epithelium, these tracers penetrate onlythrough outermost layer of cells. When applied to thesubmucosal surface, they permeate up to the top celllayers of the epithelium. While the basement membranemay present some resistance to permeation, the outerepithelium is considered to be the rate limiting step tomucosal penetration11.

There are 2 permeation pathways for passivedrug transport across the oral mucosa: paracellular

start a chain reaction of creating more free radicals, evenmore reactive and damageable from the initial one. Thechain reaction will run until the one of the antioxidantmechanisms stops it.

ROS production is inevitable in all aerobic

organisms, including humans, who necessarily posses acomplex system of antioxidant defence8,21. If homeostasisis interrupted in favour of ROS, an oxidative stresssituation is created21. Oxidative stress processes andalterations in the immune system are closely related andhave been described in different diseases, thus both theaspects also seem to be linked to the pathogenesis ofperiodontal disease, and can also be detected in plasmaof patients with periodontal disease8,16,21. However, theextent to which ROS over-generation influences theinitiation and progression of periodontal diseases is stillunknown.

The strong evidence linking ROS to the pathologicaldestruction of the connective tissue during periodontaldisease rests on the presence of neutrophil infiltrationas the main event in the host's response to bacterialinvasion1,2,10,19. The hydroxyl radical is able to initiatea classical chain reaction, known as lipid peroxidation,leading to vasodilatation and rat bone reabsorption8. Anexample of the damage caused by hydrogen peroxide isstimulation of phosphorylation of the NFkB-kB complex,activating the NK-kB and facilitating nuclear translocationand downstream of pro-inflammatory cytokines, includingIL-2, IL-6, IL-8, -interferon and TNF-, that are very

important in the pathogenesis of periodontal disease9.Experiments concerning the effects of ROS

are generally focused on the effects of gingivalinflammation. Such experiments, conducted on labanimals treated with pesticides, and given food withlowered elementary antioxidant, showed desquamationof the epithelium, swelling, elasticity loose and breakingof the collagen, bone resorption and lowered speed of Cprolin incorporation, thickening of the blood vessels andthrombosis with all its consequences5. On the contrary, inanother study, the lab animals were treated with ascorbicacid, tocopherol and biophlavonides. In this experiment,less inflammatory-destructive process was noticed12.

Mentioned data raises the questions on developingpharmaco-prophylactic measures with bio antioxidantsand other bio-regulatives as an addition to the primary andpreventive treatment of periodontal disease.

The chain of inhibition of ROS: glutathione- ascorbic acid - tocopherol, with transportation ofelectrons from pironucleotides (NAD NADF) towardsROS, guarantees permanent low level of free radicals inthe cells. Applying glutathione at periodontal disease hasproven to be effective in the early stages of the disease.Beside glutathione, other antioxidants can be used withgood results such as: tocopherol or ascorbic acid12,13.

Glutathione (GSH) is a tripeptide of glutamine,glycine, and cysteine. It is not an essential nutrient, since

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Balk J Stom, Vol 16, 2012 Application of Ascorbic Acid and Glutathione by Iontophoresis 77

The achieved results of this study speak in favour ofapplying ascorbic acid and glutathione by iontophoresisin everyday clinical practice, especially for a long termmaintenance on once achieved results of periodontaltherapy.

References

1. Chapple ILC, Brock G, Eftimiadi C, Matthews JB.Glutathione in gingival crevicular fluid and its relation tolocal antioxidant capacity in periodontal health and disease.

J Clin Pathol, 2002; 55(6):367-373.2. Chapple ILC. Role of free radicals and antioxidants in the

pathogenesis of the inflammatory periodontal diseases. JClin Pathol, 1996; 49:247-255.

3. Collins LMC.The Surface Area of the Adult Human Mouth

and Thickness of the Salivary Film Covering the Teeth andOral Mucosa.J Dent Res, 1987; 66:1300.

4. DeVries ME, Verhoef JC, J unginger HE. Developments inbuccal drug delivery. Crit Rev Ther Drug Carr Sys, 1991;8:271.

5. Enwonwu C. Cellular and molecular effects of malnutritionand their relevance to periodontal diseases. J ClinPeriodontol, 1994; 21:643-657.

6. Gandhi RE. Indirect atomic absorption determination ofatropine, diphenhydramine, tolazoline, and levamisolebased on formation of ion-associates with potassiumtetraiodometrcurate. Ind J PharmSci, 1988; 50:142.

7. Gangarosa LP, Sr. Iontophoresis in dental practice.

Chicago: Quintessence Publ Co Inc, 1983; p 40-52.8. Halliwell B, Gutteridge JMC. Free Radicals in Biology andMedicine. Oxford: Clarendon Press, 1998; pp 617-783.

9. Honda T, Domon H, Okui T, Kajita K, Amanuma R,Yamazaki K. Balance of inflammatory response in stablegingivitis and progressive periodontitis lesions. Clin ExpImmunol, 2006; 144(1):35-40.

10. Katsuragi H, Ohtake M, Kurasawa I, Saito K. Intracellularproduction and extracellular release of oxygen radicals byPMNs and oxidative stress on PMNs during phagocytosis ofperiodontopathic bacteria. Odontology, 2003; 91(1):13-18.

11. Lee JW, JHP, Robinson JR. Bioadhesive-based dosageforms: The next generation.J PharmSci, 2000; 89:850.

12. Meydani SN, Meydani M, Blumberg JB, Leka LS, SiberG, Loszewski R, Thompson C, Pedrosa MC, DiamondRD, Stollar BD. Vitamin E supplementation and in vivoimmune response in healthy elderly subjects. A randomizedcontrolled trial.JAMA, 1997; 277(17):1380-1386.

13. Nishida M, Grossi SG, Dunford RG, Ho AW, Trevisan M,Genco RJ . Dietary vitamin C and the risk for periodontaldisease.J Periodontol, 2000; 71(8):1215-1223.

14. Pandilova M, Ivanovski K, Ugrinska A, Minovska A,Radojkova V, Ristoska S. The effect of nutritional vitaminintake on periodontal health. Abstract Book of the 9thCongress of the Balkan Stomatological Society, Ohrid2004; p 97.

15. Pandilova M, Ugrinska A, Ivanovski K. Effects of nutritional

tocopherol intake on periodontal health. Mak Stom Pregl,2009; 33(1-2):95-101.

and transcellular routes4. These 2 routes can be usedsimultaneously, but 1 route is usually preferred over theother, depending on the physicochemical properties ofthe drug. Since the intercellular spaces and cytoplasmare hydrophilic in character, lipophilic compounds

would have low solubility in this environment. The cellmembrane is lipophilic in nature and hydrophilic solutionswill have difficulty permeating24.

Iontophoresis is a procedure based on the use ofgalvanic electricity and provides easy, fast and effectiveabsorption of different kinds of substances in the tissue.Iontophoresis accomplishes faster metabolism of cellsby movement of ions in the bodily fluids and opening oncanals in the cells membrane which create possibilitiesfor easer absorption of different liquid substances inthe tissue. Iontophoresis enhances drug delivery by 3mechanisms: ion-electric field interaction provides an

additional force that drives ions through the tissue, theflow of electric current increases the permeability ofthe mucosa, and electro-osmosis produces bulk motionof solvent that carries ions or neutral species with thesolvent stream. Electro-osmotic flow occurs in a varietyof membranes and is in the same direction as the flow ofcounter-ions. It may assist or hinder drug transport18,23.

Iontophoresis can also enhance mucosa delivery bya possibly dependant pore formation in the upper stratumcell layer, attributed to a flip-flop gating mechanism thatoccurs due to restructuring of the polypeptide helices onapplication of electric current. Iontophoretic transport is

capable of producing a 100 fold enhancement relative topassive diffusion7,23.

The achieved effects from our study were expected.Inflammation and gingival bleeding showed significantdecrease of the average index values for both groupsafter the therapy without noticeable difference betweenthe groups. Noticeable data were achieved for theaverage index value for gingival bleeding after 3 monthshad passed. Since our study design didnt includemethods of tracing the given medicaments into thetissue, the concentration they achieve and length of theirpermeability, the prolonged improvement of clinical

parameters may convince us that we had created betterhost environment.It is worth mentioning that during our study same

problems occurred. Convincing the patients from thestudy group to undertake the procedure was an issue,since applied at the way we did the procedure, it wasrather time consuming for both patient and doctor. Sofurther work might be focused towards developing ahydro-gel containing these or some other drugs and, usingiontophoretic enhancers, the time of appliance could beshortened.

Discoloration of the mucosa did not appear in any of

the patients, since both agents are used in dermatology asblanching agents.

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78 Maja Pandilova et al. Balk J Stom, Vol 16, 2012

22. Silness P, Loe H. Periodontal disease in pregnancy. ActaOdontol Scand, 1964; 22:121.

23. Semalty A, Semalty M, Singh R, Saraf SK, Saraf S.Iontophoretic drug delivery system: A review.Technologyand Health Care, 2007; 15(4):237-245.

24. Wertz PW. The physical, chemical and functional propertiesof lipidsin the skin. Chemistry and Physics of Lipids, 1998;91:85.

Correspondence and request for offprins to:

Pandilova MajaUniversity Dental Clinical Centre St. PanteleimonDepartment of Oral Pathology and PeriodontologySkopjeFYR MacedoniaE-mail: [email protected]

16. Panjamurthy K, Manoharan S, Ramachandran CR.Lipid peroxidation and antioxidant status in patients withperiodontitis. Cell Molec Biol Letters, 2005; 10(2):255-264.

17. Pompella A, Visvikis A, Paolicchi A, De Tata V, Casini AF.The changing faces of glutathione, a cellular protagonist.Biochemical Pharmacology, 2003; 66(8):1499-1503.

18. Rai R, Srinivas CR. Iontophoresis in dermatology. Ind JDermatol Vener Leprol, 2005; 71:236-241.

19. Sakalliolu U, Aliyev E, Eren Z, Akimek G, KeskinerI, Yavuz . Reactive oxygen species scavenging activityduring periodontal mucoperiosteal healing: an experimentalstudy in dogs. Arch Oral Biol, 2005; 50(12):1040-1046.

20. Scholz RW. Graham KS, Gumpricht E, Reddy CC.Mechanism of interaction of vitamin E and glutathione inthe protection against membrane lipid peroxidation. Ann NYAcad Sci, 1989; 570:514-517.

21. Sculley DV, Langley-Evans SC. Salivary antioxidants

and periodontal disease status. Proc Nutr Soc, 2002;

61(1):137-143.

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80 Ludmila Gavriliuc et al. Balk J Stom, Vol 16, 2012

Table 1. Fluoride concentration in drinking water in differentregions of Moldova

Regions C=mg/L Regions C=mg/L

Anenii Noi 1.7-3.0 Hincesti 2.0-5.5

Cimislia 1.78-3.2 Glodeni 2.0-8.16

Orhei 2.1-3.7 Ungheni 5.7

Floresti 3.21-3.7 Falesti 3.2-8.7

Chiadir-Lunga 2.6-4.7 Pirlita 9.0-14.0

Dental fluorosis is a developmental disturbance ofdental enamel caused by successive exposures to highconcentrations of fluoride during tooth development,leading to enamel with lower mineral content and

increased porosity. This subsurface porosity or hypo-mineralization is most likely caused by a delay inhydrolysis and removal of enamel proteins, particularlyamelogenin, as the enamel matures4.

The severity of dental fluorosis depends on whenand for how long the overexposure to fluoride occurs,the individual response, weight, physical activity,nutritional factors and bone growth5,17,20. The risk periodfor aesthetic changes in permanent teeth is between 20and 30 months of age20. The recommended level fordaily fluoride intake is 0.05-0.07 mg F/kg/day, which isconsidered of great help in preventing dental caries, actingin remineralisation. A daily intake above this safe levelleads to an increased risk of dental fluorosis. The effectsof fluoride on enamel formation suggest that fluorideaffects the enamel-forming cells, the ameloblasts3.

Fluor is the most active halogen, which iswidespread. Intoxication by fluoride leads to chronicoxidative stress and numerous pathological consequences:DNA damage, inhibition of type I collagen synthesis,altered activities of enzymes and metabolic lesions14,18.Increased free radical generation and lipid peroxidation(POL) are proposed to mediate the toxic effects offluoride on soft tissues and teeth. Oxidative stresswas evaluated by the assays of malondialdehyde and

antioxidants in patients with fluorosis. Increased POL andaltered levels of antioxidants were observed in the bloodof children with endemic skeletal fluorosis14. Many resultsare about investigation of POL and antioxidant defensesystem in human blood and urine. Data of examinationsof salivary components in patients with fluorosis are notnumerous and contradictory12,14.

Saliva, as a biological liquid of human organism,may be a reflection of the metabolic state. Salivarycomponents (indices) have clinical-diagnostic means11,16.Non-invasive methods of the salivary parametersexamination may be used in dental practice for estimation

of the degree of metabolic disturbances in patients withdental fluorosis.

The aim of this investigation was to examinecomparatively activities of glutathione-dependentenzymes, glutathione reductase and glutathione-S-transferase, and the content of glutathione in saliva ofadult patients with dental fluorosis, before and after

complex antioxidant therapy.


26 patients (19-30 years old) with mild and moderatedental fluorosis (Deans classification: 3 and 4) and 20healthy subjects (20-30 years old) were examined.

Patients were treated with complex therapy, whichincluded Opalescente Whitening gel (Ultradentproducts, USA), calcium gluconate (1.5 g/day) and

vitamins-antioxidants A (retinol palmitate -100 000 U/day), E (alfa-tocopherol acetate -100mg/day), D3 (600 U/day) and C (ascorbate - 100mg/day). The first course ofantioxidant therapy (AOT) was during 30 days, the secondAOT course was repeated 1 year after the first AOT. Thesalivary parameters (indices) were examined 3 timesduring the treatment: before the therapeutic course andtwice after the AOT processes.

Saliva was collected in the morning, before breakfast(8 am), and centrifuged at 600 g for 10 min. Aftercentrifugation saliva was examined using SP Humalyzer2000 (Germany). The contents of glutathione, protein,calcium, activities of glutathione reductase andglutathione-S-transferase were determined in saliva byspectrophotometric methods. Glutathione reductase (GR,E.C. 1.6.4.2) activity was determined with the methodalready described9. Glutathione-S-transferase (GST,E.C. 2.5.1.18) activity was determined with the methoddescribed by Habig and Jacoby10. Glutathiones content14,calcium-ions (Ca2+) content2 were determined bycorresponding methods, and protein content by pyrogallolphotometric test19. The results were statistically analyzedwith Students t-test and Microstat: Microsoft Excel 2003programme.

Results

Examination of patients with dental fluorosis showedthe sufficient difference between contents of their salivaryindices and the control group (healthy subjects). Resultsof the comparative examination of the slivery parametersare shown in figure 1. The content of protein determinedin saliva of patients with fluorosis before treatment was1.40 g/L (152.5%; p

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Balk J Stom, Vol 16, 2012 Complex Antioxidant Treatment of Dental Fluorosis 81

Figure 2. The content of glutathione (1) in saliva of patients with dentalfluorosis after complex AOT, and activities of glutathione reductase (2),

glutathione-S transferase (3). The first column - before treatment, the

second - after the 1st AOT, the third - after the 2nd AOT

(healthy subjects - 100%)

The first AOT course decreased the protein content

to 1.065 g/L (116.0%; p>0.05) and increased calcium

content to 2.042 mmol/L (85.69%; p>0.05). Thesecond AOT decreased the protein content to 1.016 g/L

(110.7%; p>0.05) and increased calcium concentrationto 2.117 mmol/L (88.8 %; p>0.05).

comparison with the healthy individuals - 240 mcmol/L).

After the first AOT, its content increased to 173 mcmol/L

(72.08%; p0.05). In saliva

of the patients with dental fluorosis the glutathione

reductase activity was 130.9 U/L (78.3%; p

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82 Ludmila Gavriliuc et al. Balk J Stom, Vol 16, 2012

human organism: moving of nerve impulse, contractionand tonus of muscles, blood clotting (the IV plasmaticfactor), stabilizer of -amylase, etc. Determination of

Ca2+-ions concentration in saliva in the patients withfluorosis showed its decreasing value. It is logical toassume that all the above-mentioned processes with Ca2+-ions participation may be changed in patients with dentalfluorosis as well6. Both courses including complex AOTand calcium gluconate increased its concentration insaliva of the patients.

Main biological role of glutathione reductase isbased on the reduction of oxidized glutathione (GSSG)to its reduced form (GSH) with utilization of NADPH+.Hydrophilic antioxidant glutathione is the maincomponent of redox-buffer system of the intracellularmedium. Glutathione-associated metabolism is a major

mechanism for cellular protection against agents whichgenerate oxidative stress and POL. Recent genetic andbiochemical evidence has demonstrated that glutathioneand glutathione-dependent enzymes play a central rolein the cellular defense against toxic agents. Enzymeglutathione-S-transferase catalyzes the conjugation ofGSH with different toxic and mutagenic compounds,which are generated during POL processes. Chronicintoxication by fluoride leads to metabolic imbalanceof antioxidant enzymes and reflects the decrease ofglutathione content and activity of glutathione reductase

in saliva of patients with dental fluorosis. Also,intoxication leads to the increase of of glutathione-S-transferase activity in saliva of the patients. Complextherapy, including AOT and calcium gluconate,increased content of glutathione and decreased activityof glutathione-S-transferase in saliva of patients withdental fluorosis. We found negative correlation betweenthe content of glutathione and clinically manifestedcharacteristics of the disease in patients with dentalfluorosis8. There was a direct relationship betweenthe activity of glutathione-S-transferase and clinical

manifestations.We found the direct correlation between clinicalcharacteristics, reflecting the level of the pathologicalprocess, and metabolic imbalance, which can allow morecorrect estimation of the intoxication degree caused byfluoride intake (Invention G2 MD, 2006)8.

Conclusions

On the basis of our results, we can confirm that

in patients with dental fluorosis an imbalance betweensalivary parameters/indices takes place as a result of

chronic intoxication with fluoride content in drinkingwater. The imbalanced salivary components - calcium,protein and glutathione-dependent enzymatic defensesystem, including glutathione, glutathione reductase andglutathione-S-transferase, were partially corrected by

complex antioxidant therapy. Moreover, 2 AOT courseswere more effective than only 1 course. In all fluoride-endemic regions of different countries it is necessary tocarry out prophylactic actions, especially laying emphasison small schools, pregnant women and feeding mothers.These prophylactic actions will decrease the risk ofchronic intoxication by fluorides.

References

1. Alvarez JA, Rezende KM, Marocho SM, Alves FB, CelibertiP, Ciamponi AL. Dental fluorosis: exposure, preventionand management. Med Oral Patol Oral Cir Bucal, 2009;14(2):E103-107.

2. Barnett RN. Photometric test, CPC method.J Clin Pathol,1973; 59:836.

3. Bronckers AL, Lyaruu DM, DenBesten PK. The impactof fluoride on ameloblasts and the mechanisms of enamelfluorosis.J Dent Res, 2009; 88(10):877-893.

4. DenBesten PK. Mechanism and timing of fluoride effectson developing enamel. J Public Health Dent, 1999;59(4):247-251.

5. Fluorides Environmental Health. Criteria 227. World Health

Organization. Geneva, 2002.6. Gavriliuc L, Stepco E, Godoroja P, Hornet V. Imbalanceof certain components of saliva patients with fluorosis.

J Oral Health Dental Management Black Sea Countries(Constanta, Romania), 2004; 3(4/10):15-19.

7. Gavriliuc LA, Stepco EA, Hornet VI, Mocan EI, CheptanaruCF, Godoroja PD. Imbalance of the biochemical parametersin saliva of patients with fluorosis. Curierul Medical(Chisinau, Moldova), 2005; 2(284):10-13.

8. Gavriliuc LA, Stepco EA, Spinei IuG, Godoroja PD. Methodof differential diagnostics of fluorosis. BOPI: MD 3163 G2,(Chisinau, Moldova), 2006; 10:32.

9. Gerasimov AM, Koroleva LA, Brusov OS, Olferev AM,Antonenkov VD, Pancenko LF. Enzymatic mechanisms ofinhibition of peroxide oxidation in different regions of ratbrain.Vopr Med Khim(Mosk), 1976; 22(1):89-94.

10. Habig WH, J acoby WB. Assays for differentiation ofglutathione S-transferase. Methods in Enzymology, 1981;77:398-405.

11. Hofman LF. Human saliva as a diagnostic specimen.J Nutr,2001; 131(5):1621-1625.

12. Huang Z, Li K, Hou G. Study on the correlation of thebiochemical indexes in fluoride workers. Zhonghua LaoDong Wei Sheng Zhi Ye Bing Za Zhi, 2002; 20(3):192-194.

13. Miao Q, Xu M, Liu B. In vivo and in vitro study on theeffect of excessive fluoride on type I collagen of rats. WeiSheng Ian Jiu, 2002; 31(3):145-147.

14. Shivarajashankara JM. Oxidative stress in children withendemic skeletal fluorosis. Fluoride, 2001; 34:103-107.

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Balk J Stom, Vol 16, 2012 Complex Antioxidant Treatment of Dental Fluorosis 83

15. Sedlak I, Lindsay RH. Estimation of total protein boundand nonprotein sulfhydryl groups in tissue with Ellmansreagents. Anal Biochem, 1968; 25(2):192-198.

16. Streckfus CF, Bigler LR. Saliva as a diagnostic fluid. OralDis, 2002; 8(2):69-76.

17. Susheela AK, Bhatnagar M. Reversal of fluoride inducedcell injury through elimination of fluoride and consumptionof diet rich in essential nutrients and antioxidants. Mol CellBiochem, 2002; 234-235(1-2):335-340.

18. Vani ML, Reddy KP. Effect of fluoride accumulation onsome enzymes of brain and gastrocnemius muscle of mice.Fluoride, 2000; 33:17-26.

19. Watanabe N, Kamei S, Ohkuto A. Urinary protein asmeasured with a pyrogallol red-molybdate complex:Manually and in a Hitachi 726 automated analyzer. ClinChem, 1986; 32:1551-1554.

20. Wong MC, Glenny AM, Tsang BW, Lo EC, Worthington

HV, Marinho VC. Topical fluoride as a cause of dental

fluorosis in children. Cochrane Database Syst Rev, 2010;

(1):CD007693.


Prof. Ludmila Gavriliuc

State University of Medicine and Pharmacy Nicolae Testemitanu

Department of Biochemistry and Clinical Biochemistry

Bul. Stefan cel Mare 165, Chisinau

MD 2004 Moldova

e-mail: [email protected]

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SUMMARYThe aim of this study was to determine the level of antimicrobial

agents Benzalkonium Chloride and Cetylpyridinium Chloride releasedfromChemFlex, a conventional glass ionomer cement (GIC). The main nullhypothesis tested was that there is no release of antimicrobial agents intothe surrounding medium.3 groups of the conventional ChemFlex GIC of

5 samples each, with BenzalconiumChloride and CPC incorporated, wereprepared - each group with a different percentage of the agents (1%, 2%,and 3%). The determination of the quantity of the antimicrobial agents wasdone by an UV- Spectrophotometer. The measurements were performed at11 successive time intervals.

The results of the statistical analysis point out that it is possible toincorporate these antimicrobial agents in conventional GIC, especially whenthe added percentage of the antimicrobial agents is 3%.

Kewords: GIC; Antimicrobial Agents; UV-Spectrophotometry

Aleksandar Dimkov, Elizabeta Gorgievska,Aleksandar Fildishevski

Faculty of Dentistry, Clinic for Pediatric andPreventive Dentistry, Skopje, FY ROM

ORIGI NAL PAPERS (OP)

Balk j Stom, 2012; 16:84-89


Release of Antimicrobial Agents fromGlass Ionomer Cements

STOMATO

LOGIC

AL

SOCIETY

Introduction

Because of the relatively frequent occurrence ofrecurrent caries after a restorative treatment, and becauseof the huge number of cariogenic microorganismsexisting in the oral cavity, which present a potentialrisk factor regarding the development of new cariouslesions, attention has increasingly been directed towardtherapeutic antimicrobial effects of restorative materials.

The glass ionomer cements (GIC) distinguishthemselves as most acceptable restorative materialspossessing the positive characteristics of fluorine in theprocesses of remineralization and antimicrobial action1-4.Their usage is determined by their characteristic to releasefluorine and to participate in the mechanism of inhibitionof the secondary caries development5-7. At the same timethey also act upon the surrounding bacteria by reducingthe cariogenic microorganisms8.

In addition to the release of fluoride ions, GICcan potentially be used as matrices for the release ofother active antimicrobial components. In regard to the

incorporation of antimicrobial agents, chlorhexidinehas been described as a golden standard for antibacterial

application9. There are several studies dealing withthe effect of incorporating chlorhexidine in differentconcentrations and its combinations in GIC to improvetheir antimicrobial properties10-14.

There are only a few data in the literature referringto the incorporation and release of other antimicrobialcomponents in GIC. Although a part of them have aconfirmed effect in the reduction of cariogenic salivaryflora when used in rinses or toothpastes, the results

regarding their incorporation in GIC are still scarce15-18.Cetylpyridinium Chloride (CPC), as an active componentof oral antiseptics, has a broad antimicrobial spectrumwith a strong bactericidal effect on Gram positivepathogens and a fungicidal effect. Its effectivenessagainst gram negative pathogens and mycobacteria isquestionable19,20. The official US pharmacopoeia acceptsBenzalkonium Chloride as an auxiliary antimicrobialagent15. It is the major antimicrobial agent in numeroustoothpastes and mouth rinses.

The objective of this study was to determine thelevel of release of the antimicrobial agents Benzalkonium

Chloride and Cetylpyridinium Chloride from theconventional GIK ChemFlex, previously incorporated

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with various concentrations of antimicrobial agents.The null hypothesis tested was that there is no releaseof antimicrobial agents into the surrounding medium,and that there are no differences due to the type ofantimicrobial agents and their represented percentages.

Materials and Methods

MaterialThe commercially available conventional GIC that

was used in the analyses was a material being widelyapplied in restorative dentistry (ChemFlex, DENTSPLYDeTrey, Konstanz, Germany). The composition of thisGIC is shown in table 1. The antimicrobial compoundsused in the study were: Cetylpyridinium Chloride (C0732

by Sigma - Aldrich Co.) and Benzalkonium Chloride(12660 by Fluka Chemical Corporation Milwaukee, WI,USA).

Table 1. The material used in the research

Material Classification Composition Manufacturer

ChemFlexConventionalglass-ionomercement

Strontium,aluminium,fluoride, silicate,tartaric acid,pigments,

polyacrylic acid

DENTSPLYDeTrey,Konstanz,Germany

3 groups of the conventional GIC ChemFlex of 5samples each, with Benzalkonium Chloride incorporated,were prepared - each group with a different percentage ofthe agent (i.e. 1%, 2% and 3%); and another 3 5 samplegroups, but with Cetylpyridinium Chloride incorporated,were also prepared - each group having the correspondingpercentage of the agent (1%, 2% and 3%).

Preparation of Samples

The antimicrobial compounds BenzalkoniumChloride (BCH) and Cetylpyridinium Chloride (CPC)were first incorporated into the GICs polyacrylic acidby mixing, and then the powder was added gradually,to the previously prepared acid and antimicrobialcompound mixture, and they were mixed together untilcomplete saturation. The antimicrobial agents wereadded in strict portions of 1, 2 and 3% of the weightof the cement. The determination of the concentration(weight) of BCH and CPC was done by measuring thegiven percentage of the antimicrobial agent with ananalytical balance. Preceding analyses had determined

the concentrations of 1, 2 and 3% of antimicrobial agentsto be equivalent to 0.0022 g, 0.0044 g and 0.0066 g, of

the whole cement mass. The freshly mixed paste wasplaced into 6 mm high metal moulds having 4 mm indiameter. The moulds had been closed by metal plateson both sides, placed in special clamps and then placedin an incubator at 37oC for 1 hour (maturation time).After their removal from the incubator, the specimens

were taken out from the clamps and moulds, and stored

individually in separate marked plastic tubes with 5 ml

de-ionized water at a temperature of 2224oC and at an air

humidity of 4050% . The sample tubes had been chosen to

allow minimal contact between the specimen and the tube

and therefore allow diffusion of antimicrobial agents from

all surfaces of the specimens into the surrounding aqueous

medium.

Determination of the Antimicrobial Agents(UV-Spectrophotometric Analysis)

The determination of the quantity of the antimicrobialagents was done by a so UV- Spectrophotometer, and theresults were expressed in Absorbance Units (AU). Thespectrophotometer was calibrated using BCH and CPCsolution with predetermined concentrations. The UVspectrophotometer was set to a detection wavelength ofmaximal absorption (214 nm) for BCH, and 259 nm forCPC.

The measurements were performed at 11 successivetime intervals as follows: 15, 30, 45 min, 1, 2, 3, 4, 24 hr,7, 14 and 30 days. During the entire period, the de-ionizedwater where the samples were stored was not changed.

The statistical analysis of the results was performedusing the 1-way ANOVA, the Post-hoc-Tukey honestsignificant difference (HSD), and the Mann-Whitney UTest.

Results

The analysis of the variances (ANOVA) showed astatistically significant difference between the averagevalues over the tested period in the group ChemFlex+ BCH both for 1%, 2% and 3%, where p=0.000000(Tab. 2). According to the post-hoc-Tukey honestsignificant difference test (HSD), the difference wasstatistically significant at 1% ChemFlex + BCH forp=0.000 between the average values at 24 hr, 7, 14 and30 days and in respect to all other average values. In thecase of 2% of the same compound, the difference wasstatistically significant for p=0.000 between the averagevalues at 2, 3, 4 and 24 hr, 7, 14 and 30 days and inrespect to all other average values, whereas in the case of3% it was statistically significant for p=0.000 between the

average values at 7, 14 and 30 days and in respect to allother average values.

Balk J Stom, Vol 16, 2012 Antimicrobial Agents Release fromGIC 85

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86 Aleksandar Dimkov et al. Balk J Stom, Vol 16, 2012

In the group ChemFlex + CPC the analysis of thevariances shows a statistically significant difference betweenthe average values over the tested period in the case of 1%CPC for p=0.000000, in the case of 2% CPC for p=0.000000and the case of 3% CPC for p=0.000103 (Tab. 3). According

to the post-hoc-Tukey honest significant difference test(HSD), when the differences in the average values were

Table 2. The average values of ChemFlex + BenzalkoniumChloride for the 3 tested concentrations over the test period(data obtained in AU)

Time

ChemFlex +Benzalkonium Chloride 1%



MEAN (SD) MEAN (SD) MEAN (SD)15 min 0.01 (0.00) 0.01 (0.00) 0.03 (0.01)

30 min 0.02 (0.00) 0.01 (0.00) 0.03 (0.01)

45 min 0.02 (0.00) 0.01 (0.00) 0.03 (0.01)

1 hour 0.02 (0.00) 0.01 (0.00) 0.04 (0.02)

2 hours 0.05 (0.01) 0.04 (0.00) 0.05 (0.01)

3 hours 0.05 (0.01) 0.04 (0.00) 0.05 (0.01)

4 hours 0.05 (0.01) 0.08 (0.00) 0.05 (0.01)

24 hours 0.07 (0.02) 0.06 (0.01) 0.07 (0.02)

7 days 0.14 (0.02) 0.16 (0.01) 0.15 (0.02)

14 days 0.12 (0.04) 0.20 (0.00) 0.17 (0.03)

30 days 0.19 (0.03) 0.25 (0.00) 0.23 (0.03)

p 0.000000 0.00 0.000000

tested individually, in the case of 1% CPC it was statisticallysignificant for p=0.000 between the average values at 7, 14and 30 days; in the case of 2% it was statistically significantfor p=0.000 between the average values at 7, 14 and 30 daysin respect to all other average values; in the case of 3% CPC,

it was statistically significant forp=0.04 between the averagevalues at 15, 30 min and 24 hr, and at 4 hr, 24 hr and 7 days.

Table 3. The average values of ChemFlex + CetylpyridiniumChloride for the 3 tested concentrations over the test period(data obtained in AU)

Time

ChemFlex +CetylpyridiniumChloride 1%



MEAN (SD) MEAN (SD) MEAN (SD)

15 min 0.01 (0.00) 0.03 (0.01) 0.06 (0.01)

30 min 0.02 (0.00) 0.02 (0.00) 0.05 (0.01)

45 min 0.02 (0.00) 0.03 (0.00) 0.06 (0.01)

1 hour 0.02 (0.00) 0.02 (0.00) 0.06 (0.01)

2 hours 0.02 (0.00) 0.03 (0.01) 0.06 (0.01)

3 hours 0.02 (0.00) 0.03 (0.01) 0.08 (0.01)

4 hours 0.02 (0.00) 0.03 (0.01) 0.09 (0.01)

24 hours 0.03 (0.00) 0.04 (0.01) 0.09 (0.01)

7 days 0.09 (0.01) 0.08 (0.00) 0.09 (0.00)

14 days 0.12 (0.02) 0.07 (0.00) 0.06 (0.01)

30 days 0.11 (0.06) 0.11 (0.01) 0.08 (0.01)

p 0.00000 0.00000 0.000103

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The finding that the specimens containing higherconcentrations of antimicrobial agent (3% in this case)released proportionally its higher amounts is in agreementwith the results of Ribeiro and Ericson21. In contrast to

our study the GIC used were ChemFil and AquaCem. Theconcentrations of the antimicrobial agents used were alsodifferent (7.5% and 8.2% w/w) chlorhexidine digluconate,respectively, and 13.3% w/w of chlorhexidine diacetate. Incontrast to our study, the specimens used by the authorsof the aforementioned study had far lower dimensions(3 mm diameter and 1.5 mm thickness). Chlorhexidinedigluconate was quickly released from AquaCem and after5 days no measurable increase of concentration was seen.This tendency was also seen with diacetate /AquaCem.

Similar to the previously mentioned results are thoseobtained by investigating the use of an experimental GIC

as a carrier for the release of chlorhexidine acetate21

.The concentrations range was from 0.5% to 13.0% of

Table 4. Mann-Whitney U Test for the average values between groups treated with ChemFlex + BenzalkoniumChloride and

ChemFlex + Cetylpyridiniun Chloride

Time

ChemFlex +BCH 1%ChemFlex +CPC 1%



p-level p-level p-level

15 min 0.174526 0.016294* 0.047203*

30 min 0.676104 0.009024* 0.047203*

45 min 0.250593 0.009024* 0.047203*

1 hour 0.117186 0.174526 0.047203*

2 hours 0.009024* 0.174526 0.117186

3 hours 0.016294* 0.174526 0.075801

4 hours 0.009024* 0.009024* 0.028281*

24 hours 0.009024* 0.075801 0.117186

7 days 0.016294* 0.009024* 0.009024*14 days 0.464703 0.009024* 0.009024*

30 days 0.016294 0.009024* 0.009024*

* - Statistical significant

The difference in the average values obtained bytreatment with ChemFlex +BCH 1% and ChemFlex +CPC 1% was statistically significant between the valuesat 2, 3, 4, 24 hours and 7 days for p0.05

(Tab. 4). The difference in the average values obtainedby treatment with ChemFlex +BCH 2% and ChemFlex +CPC 2% was statistically significant between the values

at 15, 30, 45 min, 4 hr and 7,14 and 30 days for p0.05. The difference in the average values obtainedby treatment with ChemFlex +BCH 3% and ChemFlex+ CPC 3% was statistically significant between the

values at 15, 30, 45 min, 1 and 4 hr and 7, 14 and 30days for p0.05.

Discussion

This study is a part of a comprehensive studydealing with the possibility to incorporate antimicrobial

components from the group of quaternary ammoniumcompounds in conventional GIC.

The results show a continual release of theantimicrobial compounds, starting at 15th minute, witha tendency of increase at all periods of measurement.The highest values were obtained at the end of the testtime period, i.e. after one month, and for the highesttested concentration - 3%. The conclusion drawn fromthe statistical analysis stresses that the addition of BCHand CPC shows highly significant differences betweenthe average values over the tested period and betweenthe different percentages, and the incorporation of 3%

antimicrobial components into GIC as being the mostappropriate.

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88 Aleksandar Dimkov et al. Balk J Stom, Vol 16, 2012

chlorhexidine acetate by weight. In contrast to our study,release into water was examined using high-performanceliquid chromatography. The specimens had an internaldiameter of 10 mm and a thickness of 1 mm. In thiscase the release of the antimicrobial compound had also

occurred relatively soon, all measurable chlorhexidinewas released within 22 hr; however, that was less than10% of the total mass incorporated in the specimens.

In both quoted papers, incorporation of theantimicrobial compound was made in the GIC powder.Contrary to this, in this study, incorporation of the 2antimicrobial compounds was in the GIC liquid, becauseof the chemical structure of the antimicrobial compoundsand the better distribution of the long chains of thequaternary compounds in a liquid medium. The data thatan increased percentage of incorporated chlorhexidineacetate gave an increased release into the water is in

correspondence with our results. The increasing amountsof incorporated antimicrobial agents (chlorhexidinediacetate) did not result in significant increases in elutedconcentrations, which is in contrast to our study and alsoin contrast to the studies mentioned above14.

Attempts have been made to incorporateantimicrobial compounds in other restorative materials byinvestigating the ability of incorporation of antimicrobialagent CPC in the resin matrix22. Among the antimicrobialproperties of the resin, the releasing of CPC into water wasinvestigated as well, using a UV-vis spectrophotometerduring the different time periods. The results revealed thatless than 0.11 AU of CPC was released into water for allspecimens. In our case, the release of CPC in water in theamount of 0.11 AU+/0.2 AU was noted on the 14th and30th day on an average in 1% ChemFlex +CPC; on the30th day on the average for 2%, and on the 30th day in onespecimen for 3% ChemFlex +CPC.

As for this study, there is a question of thecumulative effect of the compounds in the medium(de-ionized water). The increase of the level of releasedcompounds increase with time, which speaks of additionaldilutions of the compounds in the medium. In orderto see the anticariogenic effect of the compounds onthe oral flora, i.e. in order to make a parallel with the

effect that would have occurred inside the oral cavity, itwould be necessary to perform certain microbiologicalanalyses as well. Even though the majority of studiesanalyze the release of the compounds in a fresh mediumbefore each measurement, the cumulative effect of theleached antimicrobial compounds is very important,above all in the cases of poor oral hygiene. An aspect ofno less importance, yet, is the effect that such modifiedGIC could have towards the cavities in the course ofapplication, i.e. the effect of the antimicrobial compoundsbeing deposited at the junctures between the restorationand the dentine, and their protective effect. In situations

of implementing a sound oral hygiene, the cumulativeeffect of antimicrobial compounds would not be of such

importance because of frequent rinsing of the oral cavity.Regarding such cases, an analysis should be made of therelease of antimicrobial compounds in the medium bychanging the latter in predefined time intervals.

Conclusions

1. The addition of BCH and CPC shows highsignificant differences between the average valuesover the tested period and between the differentpercentages.

2. The incorporation of 3% antimicrobial components(BCH and CPC) into GIC is the most appropriate.

Acknowledgments: This article was submitted in

partial fulfilment of a PhD degree at the School ofScience, University of Greenwich, UK and the Facultyof Dentistry - Skopje, FYROM. I should like to thank toProfessor John W. Nicholson for the award of a VisitingFellowship which allowed complete experimental workreported in this paper to be carried out at the University ofGreenwich, UK.

References

3. McLean JW, Nicholson JW, Wilson AD. Proposednomenclature for glass-ionomer dental cements and relatedmaterials. Quintessence Int, 1994; 25:587-589.

4. Nicholson JW. Chemistry of glass-ionomer cements: areview. Biomaterials, 1998; 19:485-494.

5. Nicholson JW. Adhesive dental materials and theirdurability. Int J Adhesion Adhesives, 2000; 20:11-16.

6. Attar N, Onen A. Fluoride release and uptake characteristicsof aesthetic restorative materials. J Oral Rehab, 2002;29:791-798.

7. Berg JH. The continuum of restorative materials in pediatricdentistry - a review for the clinician. Pediatric Dentistry,1998; 20:93-100.

8. Dionysopoulos P, Kotsanos N, Pataridou A. Fluoride releaseand uptake by four new fluoride releasing restorativematerials.J Oral Rehab, 2003; 30:866-872.

9. Billington RW, Williams JA, Dorban A, Pearson GJ . Glassionomer cement: evidence pointing to fluorine release in theform of monofluorophosphate in addition to fluoride ion.Biomaterials, 2004; 25:3399-3402.

10. Hengtrakool C, Pearson GJ , Wilson M. Interaction betweenGIC and S. sanguis biofilms: Antibacterial properties andchanges of surface hardness.J Dentistry, 2006; 34:588-597.

11. Leunga D, Spratt DA, Pratten J, Gulabivala K, Mordan NJ ,Young MN. Chlorhexidine-releasing methacrylate dentalcomposite materials. Biomaterials, 2005; 26:7145-7153.

12. J edrychowski J R, Caputo AA, Kerper S. Antibacterial and

mechanical properties of restorative materials combinedwith chlorhexidine.J Oral Rehabil, 1983; 10:373-381.

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13. Patel MP, Cruchley AT, Coleman DC, Swai H, Braden M,Williams DM. A polymeric system for the intra-oral deliveryof an anti-fungal agent. Biomaterials, 2001; 22:2319-2324.

14. Sanders BJ, Gregory RL, Moore K, Avery DR.Antibacterial and physical properties of resin modifiedglass-ionomers combined with chlorhexidine. J OralRehab, 2002; 29:553-558.

15. Botelho MG. Inhibitory effects on selected oral bacteria ofantibacterial agents incorporated in glass ionomer cements.Caries Research, 2003; 7:108-114.

16. Takahashi Y, Imazato S, Kaneshiro AV, Ebisu S, FrenckenJ E, Tay FR. Antibacterial effects and physical propertiesof glass-ionomer cements containing chlorhexidine for theART approach. Dent Mater, 2006; 22:647-652.

17. Block, SS. Disinfection, Sterilization and Preservation.Fourth Edition. Philadelphia - London: Lea & Febiger,1991; pp 225-242, 274-286.

18. Ciancio S. Expanded and future uses of mouth rinses.J AmDent Assoc, 1994; 25(Suppl 2):29S-32S.

19. DePaola LG, Minah GE, Overholser CD, Meiller TF,Charles CH, Harper DS, McAlary M. Effect of anantiseptic mouth rinse on salivary microbiota. AmJ Dent,

1996; 9: 93-95.

20. Charles CH, Sharma NC, Galustians HJ , Qaqish J ,McGuire JA, Vincent JW. Comparative efficacy of anantiseptic mouth rinse and an antiplaque/antigingivitisdentifrice. A six-month clinical trial. J Am Dent Assoc,2001; 32:670-675.

21. Radford JR, Beighton D, Nugent Z, Jackson RJ . Effect ofuse of 0.05% Cetylpyridinium Chloride mouthwash onnormal oral flora.J Dent, 1997; 25:35-40.

22. Pitten FA, Kramer A. Efficacy of Cetylpyridinium Chlorideused as oropharingeal antiseptic. Arzneimittelforschung,

2001; 51:588-595.23. Ribeiro J , Ericson D. In vitro antibacterial effect of

chlorhexidine added to glass-ionomer cements. Scand JDent Res, 1991; 99:533-540.

24. Palmer G, Jones FH, Billington RW, Pearson GJ .Chlorhexidine release from an experimental glass ionomercement.Biomaterials, 2004; 25:5423-5431.

25. Namaba N, Yoshida Y, Nagaoka N, Takashima S, YoshimotoKM, Maeda H, van Meerbeek B. Antibacterial effect ofbactericide immobilized in resin matrix. Dental Mat, 2009;25:424-430.


Aleksandar DimkovFaculty of Dentistry, Clinic for Pediatric and Preventive DentistrySkopje, FY R MacedoniaE-mail: [email protected]

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SUMMARYCurved root canals are challenge for instrumentation, preparation,

irrigation and obturation. The aim of the present study was to find theworking length (WL) and irrigation efficiency in root canals with curvatures30-45 and in root canals with anatomical abnormalities 45-90.

68 human, matured, extracted molars with 201 root canals were

included in the study. Molars were placed in 3 groups in relation to theangle between the root and the axis. The first group were teeth with straightcanals (25-30, a control group; 14 teeth - 45 root canals), the secondgroup were teeth with curved canals (30-45; 22 teeth 66 root canals) andthird group were teeth with severely curved canals (45-90; 31 teeth with96 root canals). Measurements: mesio-distal buccal size of the chamber inits largest part and both bucco-lingual sizes - mesial as L1 and distal as L2.Root canal preparation: removal of the root pulp with K endofiles number6, 8 and 10, with Step-Down and Balance-force techniques. Canals lengthwas measured radiographically by intra-oral radiographs, preparationcontinuous after X-ray analysis of the level of penetration of irrigants withcontrast solution of diluted Urograffin 66%. Regime of active irrigation:same for all groups with 2% H2O2 and 1% NaOCl and paper points drying.

To follow up the results a fourth radiograph was made and a second onewith Urograffin. The applied criteria for WL and for penetration of theirrigant was as follows: 3 - the whole WL; 2 - 1 mmshorter than WL; 1 - 2mmshorter than WL; and 0 - more than 2 mmshorter than WL.

The active irrigation was more efficient in curved root canals, becausein straight canals most of the irrigant was lost, back in the mouth orperiapically. In straight root canals, only moisturizing (Miller pins) of thecanal can be effective and less dangerous.

Kewords: Endodontics; Working Length; Root Canals, curved

Ekaterina Boteva1, Dimitar yovchev2

Faculty of Dental Medicine, Sofia, Bulgaria1Department of Conservative Dentistry2Department of Imaging and Oral Diagnostics

ORIGI NAL PAPER (OP)

Balk j Stom, 2012; 16:90-93


Efficacy of Working Length Detection andIrrigation during Preparation of Curved Root Canals

STOMATO

LOGIC

AL

SOCIETY

Introduction

Curved root canals are well known challenge forinstrumentation, preparation, irrigation and obturation.Root curvatures with abnormalities over 45 are notinvestigated from this point of view at all. Iatrogenicerrors are often associated with these teeth. In the last15 years, between 1995 and 2010, only 13 articles arerelated to this problem, excluding those with extrememethodology as the use of 6.25% NaOCl.

All published articles are researches from in vitro

studies and surprisingly the curvatures are from 20 upto 40. Teeth with curvatures between 25 and 45, as we

found in our previous studies, are from 16-19% of allteeth, and those with curvature of 45 are more than 1.3%.The number of experimental teeth in most of the studiesvaries between 30 and 135, while the average numbersare 59-621-14. In most studies, the preparation techniquewas reported to be Step-Down1,2,5-9,11,12. Differentinstrumentations with hand files6,13 and machine rotaryfiles10,11,14 were used.

Apical preparation size varied from 10-25 to

40-45, inrelation tothedegreeof curvature.In these

papers, the differences are not only in the irrigation

regimes but in type of medication. NaOCl was used as2.5-5%3,7,10 and in combination with EDTA5,6. In all

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H22 was used for irrigation. The followed up methodswere x-ray3, SEM8, bioluminiscense7,9, intra-operativemicroscopy2, stereomicroscopy13 and light microscopy6.

In most of the cases, the results were predictable andmostly a logical result from the design of the experiment.

Summarizing the results, it can be concluded that the typeof preparation is a major factor for the degree of removalof the smear layer and penetration of irrigants andmedicines. The highest efficiency was 70% in one groupin one article. In most articles, penetration of irrigants wasnot complete among curvatures between 30-40.

The aim of the present study was to find theworking length and irrigation efficacy in root canalswith curvatures 30-45 and in those with anatomicalabnormalities 45-90.


Teeth: 68 human matured extracted molars with 201root canals are included in the study. All teeth are from thesame geographical region.

Groups: Molars are separated in 3 groups in relationto the angle between the root and the axis. Molars withstraight roots (25-30) were a control group (14 teeth with45 root canals), the second group were molars with curvedroots (30-45; 22 teeth with 66 root canals), and the thirdgroup were teeth with severely curved roots (45-90; 31

teeth with 96 root canals).Measurements: Mesio-distal buccal size of the

chamber in its largest part and both bucco-lingual sizes- mesial as L1 and distal like L2, and the average of lasttwo as L.

Root canal preparation: Started with opening theorifices with manual Orifice Openers and removal of theroot pulp with K endofiles number 6, 8 and 10, usingStep-Down and Balance force techniques. Root canallength was measured radiographically with K files, andpreparation continued after X-ray analysis of the level ofpenetration of irrigants with contrast solution of diluted

Urograffin 66%.Regime of active irrigation: It was the same for all

groups - with 2% H2O2 and 1% NaOCl and paper pointsdrying. The aim of root canal preparation was even inroots with 90 curvatures the apical stop to be number20-25, and for the rest of the teeth 30-40. To

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