Serbian Dental J, 2007, COBISS. SR-ID UDC: : ISSN DOI: 10.

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1 Serbian Dental J, 2007, UDC: : ISSN Histološke promene u parodoncijumu primenom limunske kiseline u terapiji parodontopatije Histological changes in periodontal tissue caused by the application of citric acid in the treatment of periodontal disease COBISS. SR-ID DOI: /SGS N Ružica Nedeljkovi 1, Petar Boji 2, Obrad Zeli 3 1 Klinika za bolesti zuba, Stomatološki fakultet u Beogradu 2 Institut za patologiju, Stomatološki fakultet u Beogradu 3 Klinika za parodontologiju i oralnu medicinu, Stomatološki fakultet u Beogradu 1 Dept. of Restorative Dentistry and Endodontics, School of Dentistry, Belgrade 2 Institute for Pathology, School of Dentistry, Belgrade 3 Dept. of Periodontology and Oral Medicine, School of Dentistry, Belgrade ORIGINALNI RAD (OR) ORIGINAL ARTICLE KRATAK SADRŽAJ Uvod: Kondicioniranje površina parodontopati nih zuba u hirurškoj terapiji parodontopatije ima zna ajnu ulogu u reparaciji obolelog parodoncijuma. Cilj ovog istraživanja je bio da se histološki proveri uticaj limunske kiseline na indukciju vezivno tkivnog pripoja, odnosno na cementogenezu i resorpciju cementa i dentina posle repozicije režnja na denudiranim površinama zuba. Materijal i metod: Istraživanje je obavljeno na svinjama rase beli Landras. Eksperiment je obavljen na 12 životinja, 6 ženskog i 6 muškog pola sa zastupljenom mle nom denticijom. U cilju što adekvatnije procene dobijenih rezultata i pore enja koris ena je tehnika podeljenih usta. Denudirane površine donjih o njaka sa leve strane su tretirane lateralno pomerenim režnjem (LPR), a ogoljene površine istih zuba sa desne strane prekrivane slobodnim mukogingivalnim autotransplantaima (SMAT). Ekscizijom tkiva oko denudiranih površina osveženo je i pripremljeno mesto za prijem režnja, a površina zuba obra ena ru nim instrumentima. Kao donor mesto za operaciju LPR koriš en je bezubi region lateralno od tretiranog zuba, gde je odignut mukoperiostalni režanj dovoljne veli ine da potpuno prekrije ogoljenu površinu zuba preko gle no cementne granice. Eksperimentalnoj grupi zivotinja je pre repozicije režnja sterilnom kuglicom vate apli- SUMMARY Introduction: Surface conditioning of teeth with periodontal disease in the surgical treatment of periodontal disease has an important role in the reparation of periodontal tissue. The aim of this study was to evaluate histologically the effect of citric acid on the induction of connective tissue attachment, i.e. cementogenesis and the resorption of cementum and dentin after flap reposition on the exposed tooth surfaces. Materials and Methods: The study was conducted on 12 White Landras pigs, 6 male and 6 female, with deciduous dentition. The split mouth technique was used for more adequate assessment and comparison of data. Exposed surfaces of the upper left canine were treated with laterally positioned flap (LPF) and their counterparts with free gingival graft (FGG). The recipient site was prepared by tissue excision and the tooth surface was prepared using hand instruments. The donor site for LPF was the edentulous region lateral to the treated tooth, where the full width flap was lifted to completely cover the exposed root surface over the enamel-cementum junction. In the experimental group, a fresh solution of citric acid (ph=1) was applied for 3-5 min with a sterile cotton pellet prior to the flap reposition. The tooth was rinsed with saline and the wound was sutured with single sutures. In

2 212 Stom Glas S, vol. 54, kovan svež rastvor koncentrovane limunske kiseline ( ph1) u trajanju od 3-5 min. zub je ispran fiziološkim rastvorom, a rana zašivena pojedina nim šavovima. Kontrolnoj grupa životinja je ura ena identi na hirurska intervencija bez primene limunske kiseline. SMAT odnosno režanj poludebnjine, koriš en za prekrivanje denudiranih površina zuba uzet je sa bezubog alveolarnog grebena lateralno od tretiranih zuba. Pre postavljanja transplantata eksperimentalnoj grupi životinja je aplikovana limunska kiselinu na isti na in kao i životinjama tretiranih LPR. Transplantatom je prekrivena ogoljena površina korena. Fiksacija je ura ena pojedina nim šavovima, a rana zašti ena hirurškim pakovanjem. Konrolna grupa životinja je tretirana bez limunske kiseline. Životinje su žrtvovane osam nedelja nakon primene navedenih mukogingivalnih hirurških zahvata. Uzeti materijal je obra en rutinskim postupkom koji je predvi- en za histološka ispitivanja tkiva svetlosnom mikroskopijom. Posle fiksacije u 10% formalinu, materijal je stajao u dekalcinatu 7-10 dana. Parafinski kalupljien materijal je se en na preparate debljine oko 5 mikrometara koji su nakon fiksacije na mikroskopskim plo icama bojeni hemotoksin eozinom (HE), Masson- Trihrom -om Paf- Hallmi-em. Stanje parodontalnih tkiva eksperimentalnih životinja analizirano je svetlosnom mikroskopijom. Rezultati: Eksperimentalno istraživanje je pokazalo da limunska kiselina aplikovana na ogoljenu površinu zuba u toku režanj operacije ima pozitivan uticaj na cementogenezu. Na površini korena duž cementa evidentno je prisustvo cementoblasta postavljenih gusto i u nizu. Novoformirani cement je hiperplasti an i pravilno slojevite gra e, evidentno vise prisutan na modelima životinja tretiranih režnjem pune debljine. Nije registrovano ošte enje odontoblasta i vezivnog tkiva pulpe. Klju ne re i: ogoljena površina zuba, limunska kiselina, lateralno pomereni režanj, slobodni mukogingivalni autotransplantat the control group, the identical surgical treatment was performed but without citric acid. FGG, i.e. half width flap taken from the edentulous region lateral to the treated teeth, was used to cover the exposed tooth surfaces. Prior to FGG positioning in the experimental group, citric acid was applied in the same manner as in animals treated with LPF. The graft covered the exposed root surface. Fixation was done with single sutures and the wound was additionally preserved with surgical dressing. The control group was treated without citric acid. The animals were sacrificed eight weeks after the treatment. The material was prepared in a routine manner for histological sampling under light microscopy. After 10% formalin fixation, the material was decalcified for 7-10 days. Paraffin molds were cut in 5 m thick slices which were stained with haematoxylin eosin (HE), Masson-Trichrome and Paf-Hallmi. Periodontal tissue status in experimental animals was analyzed under the light microscope. Results: Citric acid applied on the exposed tooth surface during the flap surgery had a positive effect on cementogenesis. Cementoblasts were observed along root cementum in thick rows. Newly formed cementum was hyperplastic, with regular laminar structure and more evident on animal models treated with full width flap. No damage of odontoblasts or pulpal connective tissue was observed. Keywords: exposed tooth surface, citric acid, laterally positioned flap, free gingival graft. Upotrebu kiselina za demineralizaciju korenova parodontopati nih zuba i formiranje novog pripoja su još s kraja XIX veka prvi opisali Yunger i Steward. Oni su uo ili da se demineralizacijom obolelih korenova stimulišu induktivni potencijal korena zuba, alveolarne kosti i mekog tkiva 1. Urist 2 je potvrdio da demineralizovani dentin implantiran intramuskularno indukuje formiranje kosti. Mezenhimalne elije koje dolaze u kontakt sa demineralizovanim dentinskim matriksom mogu stvoriti povoljne uslove za koštanu morfogenezu. Nove elije sposobne za osteogenezu se formiraju izme u l0- og i 15- og dana, a kost ak 30-og dana posle implantacije. Dalja istraživanja u ovom smislu nastavili su Register i Burdick 3 sa ciljem da ispitaju optimalne mogu nosti primene limunske kiseline, odnosno njene koncentracije i vremena aplikacije potrebne da indukuje reparatorni potencijal kolagenih Acid application for root demineralization in teeth with periodontal disease and the formation of a new gingival attachment was described by Yunger and Steward in late 19 th century. They observed that root demineralization stimulated the inductive potential of the root, alveolar bone and soft tissue 1. Urist 2 confirmed that demineralized dentin, implanted intramuscularly, induced new bone formation. Mesenchymal cells in contact with demineralized dentin matrix may create favorable conditions for bone morphogenesis. New cells capable of osteogenesis are formed between day 10 and 15 whereas the bone is formed on day 30 after implantation. Further studies in this field were conducted by Register and Burdick 3 with the aim to investigate optimal possibilities for the application of citric acid, its concentration and time, in order

3 Serbian Dental J, 2007, to induce the reparative potential of collagen fibrils, i.e. connective tissue of periodontium and cementum. Further studies on animals were aimed at investigating the effect of demineralized cementum in the induction of new attachment. It was reported that demineralization may reduce the resorptive phase after surgery by enhancing cementogenesis and new attachment formation. Register surgically treated periodontal pockets and applied citric acid for 2 min on exposed root surfaces. Histologically and histometrically, he proved the formation of new attached epithelium and connective tissue 7 mm in length. Histological investigations by Register and Burdick published a year later confirmed that tiny cementum tags were deposited in clean and wide demineralized dentinal tubules, mechanically supporting the new attachment. Studies associated with citric acid were continued by Stahl and Froum, Garret, Crigger and Egelbourg, Morrison and Thompson 1. It was proved in vitro that citric acid had the ability to dissolve calcium phosphate from the bone and decalcify enamel and dentin. Neuman et al. 1 tested three hypotheses about the dissolution of hydroxyapatite crystals in vivo. Hydrogen ions had an important ability to demineralize crystal structures. The second hypothesis was based on the ability of a chelating agent to remove calcium from hydroxyapatite crystals and the third on the fact that certain ions were involved in surface exchange with hydroxyapatite crystal. Citric acid has the threefold effect on hydroxyapatite: releases ions as a mild acid, reacts with the chelating effect of calcium with 2 or 3 molecular groups and its ions may exchange phosphate ions in hydroxyapatite by surface exchange. TEM studies showed that acid applied on exposed root surfaces of teeth with periodontal disease, not treated causally, had no effect. Isckaw et al. 1 found that peritubular dentin dissolved primarily in chronically exposed dentin treated with unspecific acids, unlike peritubular dentin in abraded or eroded root surfaces. Based on studies by McKay, Steacher, Sobel, Gallin, Polson and Proy 1, the effect of citric acid in the induction of new attachment can be explained. Studying human material, Polson and Proy proved a special relationship between demineralized dentin and connective tissue; exposed collagen fibrils of demineralized dentin attached to the collagen fibers of the adjacent tissue. These fibrils had a specific, functional orientation as chain links, presenting a strong mechanical barrier and preventing apical migration of epithelial cells from the surface as well as haemotactic stimuli. In this way, apical migration of attached epithelium was prevented, creating favorable conditions for further organization of the connective tissue attachment, increase in collagen fibrils and cementogenesis. Citric acid induced cementogenesis by activating cementoblast and osteoblast inductors as well as deactifibrila, odnosno vezivnog tkiva periodoncijuma i cementa. Kasnija istraživanja obavljena na eksperimentalnim životinjama imala su za cilj da ispitaju sposobnost demineralizovanog cementa da indukuje novi pripoj. Utvr eno je da demineralizacija moze da smanji resorptivnu fazu posle operacije tako sto ubzava cementogenezu i formiranje novog pripoja. Register je hirurški tretirao parodontalne džepove, a obra ene površine ogoljenih korenova tretirao koncentrovanom limunskom kiselinom u trajanju od 2 min. Histološki i histometrijski je dokazao formiranje novog pripojnog epitela i veziva prose ne dužine 7mm. Histološka ispitivanja Resister- a i Burdick-a obavljena godinu dan kasnije su pokazala da se u o iš enim i proširenim tubulima demineralizovanog dantina deponuju igli- asti produžeci cementa što predsavlja mehani ku potporu za formiranje novog pripoja. Istraživanja vezana za limunsku kiselinu nastavili su Stahl i Froum, Garret, Crigger i Egelbourg Morrison i Thompson 1. Dokazano je in vitro da limunska kiselina ima sposobnost rastvaranja kalcijum fosfata iz kosti, i dekalcifikacije gle i i dentina. Neuman i sar. 1 su postavili tri hipoteze o rastvaranju kristala hidroksil apatita in vivo. Zna ajna je sposobnost hidrogenih jona da demineralizuju kristalne strukture. Druga hipoteza bazira na sposobnosti helatnog agensa da izvla i kalcijum iz kristala hidroksil apatita, a tre a pretpostavka je zasnovana na injenici da izvesni joni u estvuju u površinskoj razmeni sa hidroksil apatitnim kristalom. Limunska kiselina deluje na hidroksil apatit na sva tri na ina; osloba a jone kao slaba kiselina, deluje na helatni agens kalcijuma sa dve ili više grupa u svom molekulu i joni limunske kiseline mogu površinskom razmenom da zamene jone fosfata u hidroksil apatitu. Ispitivanja transmisionom electron mikroskopijom su pokazala da kiselina aplikovana regionalno na ogoljene površine korena parodontopati nih zuba, ali koji nisu tretirani parodontološki, nema nikakvog efekta. Isckaw-a i sar. 1 su utvrdili da se na hroni no eksponiranom dentinu tretiranom nespecifi nim kiselinama peritubularni dentin prioritetno rastvara u odnosu na peritubularni dentin abradirane ili erodirane površine korena. Na osnovu ispitivanja Mc Kay-a, Steacher-a, Sobel-a, Gallin-a, Polson-a i Proy-a 1 može se objasniti uloga limunske kiseline u indukciji novog pripoja. Polson i Proy su radovima na humanom materijalu dokazali poseban tip veze izme u demineralizovanog dentina i vezivnog tkiva; eksponirane kolagene fibrile demineralizovanog dentina se spajaju sa kolagenim vlaknima susednog tkiva. Ova fibrinska vlakna imaju specifi nu, funcionalnu orijentaciju kao karike na lancu što predstavlja jaku mehani ku prepreku i onemogu ava apikalnu migraciju epitelnih elija sa površine, a istovremeno je i odbrambena barijera od hemotakti kih nadražaja. Na taj na in se zaustavlja apikalna migracija pripojnog epitela i stvaraju povoljni uslovi za dalju organizaciju pripojnog vezivnog tkiva, umnožavanje kolagenih fibrila i kasnije cementogenezu. Limunska kiselina indukuje cementogenezu aktiviranjem induktora

4 214 Stom Glas S, vol. 54, cementoblasta i osteoblasta kao i deaktiviranjem inhibitora regeneracije. Po etno formiranje cementa je registrovano izmedu 42 i 60 dana od operacije i to u koronarnom delu 1. Cilj ovog istraživanja je bio da se histološki proveri uticaj limunske kiseline na indukciju vezivno tkivnog pripoja, odnosno na cementogenezu i resorpciju cementa i dentina posle repozicije režnja na denudiranim površinama zuba. vating the inhibitors of regeneration. An initial cementum formation was observed in the coronal area between day 42 and 60 after surgery 1. The aim of this study was to evaluate histologically the effect of citric acid on the induction of connective tissue attachment, i.e. cementogenesis and the resorption of cementum and dentin after flap reposition on the exposed tooth surfaces. Materijal i metod Istraživanje je obavljeno na svinjama rase beli Landras. Eksperiment je obavljen na 12 životinja, 6 ženskog i 6 muškog pola sa zastupljenom mle nom denticijom. Na po etku rada životinje su bile stare 2 meseca i prose no teske oko 20 kg. U toku eksperimenta jedna životinja je uginula. Operacije lateralno pomerenog režnja (LPR) i transplantacije su ra ene u predelu donje vilice na o njacima bilateralno. Odabiranjem životinja iz istog legla obezbe ena je homogenost uzorka u odnosu na rasu, pol, starost, stanje parodontalnog tkiva i na in ishrane. U cilju što adekvatnije procene dobijenih rezultata i pore enja koriš ena je tehnika podeljenih usta. Denudirane površine donjih o njaka sa leve strane su tretirane lateralno pomerenim režnjem (LPR), a ogoljene površine istih zuba sa desne strane prekrivane slobodnim mukogingivalnim autotransplantatima (SMAT). LPR je koriš en kao mukoperiostalni režanj što je omogu ilo ispitivanje reate mena tkiva nastalog repozicijom režnja pune debljine u eksperimentalnoj i kontrolnoj grupi. SMAT-om je histološki pra en novoformirani pripoj posle repozicije režnja poludebljine u obe grupe životinja. Svi hirurški zahvati na eksperimentalnim životinjama su ra eni u intravenoznoj anesteziji. U pripremnoj fazi eksperimenta na o njacima donje vilice su pravljeni arteficijalni defekti gingive sli ni onima u parodontopatiji. Da bi se spre ila epitelizacija rane oko zuba postavljana je ži ana ligatura sa slobodnim krajem na denudiranoj površini zuba. Ovako ogoljene površine zuba koje su bile izložene uticaju pljuva ke nakon 8 nedelja su prekrivane sa LPR ili SMAT-om. LPR-om su prekrivane ogoljene površine o njaka u donjoj vilici sa leve strane. Ekscizijom tkiva oko denudirane površine osveženo je i pripremljeno mesto za prijem režnja, a površina zuba obra ena ru nim instrumentima. Kao donor mesto koriš en je bezubi region lateralno od tretiranog zuba, gde je odignut mukoperiostalni režanj dovoljne veli ine da potpuno prekrije ogoljenu površinu zuba preko gle no-cementne granice. Eksperimentalnoj grupi životinja je sterilnom kuglicom vate aplikovan svež rastvor koncentrovane limunske kiseline ( ph1) u trajanju od 3-5 min posle ega je zub ispran fiziološkim rastvorom, rana posušena i prekrivena reponovanim mukoperiostalnim režnjem koji je pojedina nim šavovima fiksiran u novom lateralnnom položaju. Kontrol- Materials and Methods The study was conducted on 12 White Landras pigs, 6 male and 6 female, with deciduous dentition. At the beginning of the study, the animals were 2 months old, with the average weight of 20 kg. During the experiment, one animal died. Laterally positioned flap (LPF) and free gingival graft (FGG) surgery was performed on lower canines, bilaterally. Taking the animals from the same breed, we assured sample homogeneity with respect to race, gender, age, periodontal status and feeding habits. The split mouth technique was used for more adequate assessment and comparison of data. Exposed surfaces of the upper left canine were treated with LPF and their counterparts with FGG. LPF was used as the mucoperiosteal flap which enabled the comparison of tissue reattachment created by full width flap reposition in experimental and control groups. FGG was used to histologically evaluate the newly formed attachment after half width flap reposition in both animal groups. All surgical treatments were conducted in intravenous anesthesia. In the preliminary phase of the experiment, artificial gingival defects similar to those in periodontal disease were created on lower canines. In order to prevent wound epithelization around the teeth, a wire ligature was attached to the exposed tooth surface. These tooth surfaces were exposed to saliva for 8 weeks and covered with either LPF or FGG. LPF was used to cover the exposed canine surface on the left side. The recipient site was prepared by tissue excision and the tooth surface was prepared using hand instruments. The donor site for LPF was the edentulous region lateral to the treated tooth, where the full width flap was lifted to completely cover the exposed root surface over the enamel-cementum junction. In the experimental group, a fresh solution of citric acid (ph=1) was applied for 3-5 min with a sterile cotton pellet prior to the flap reposition. The tooth was rinsed with saline and the wound was sutured with single sutures in the new lateral

5 Serbian Dental J, 2007, na grupa životinja nije tretirana limunskom kiselinom. Denudirane površine u donjoj vilici sa desne strane su prekrivane SMAT-om, odnosno režnjem poludebnjine. Ekscizijom mekog tkiva oko denudacije je pripremljeno mesto za prijem režnja, a ogoljene površine su pripremane na isti na in.transplantat odnosno režanj poludebljine je uzet sa bezubog alveolarnog grebena lateralno od tretiranog zuba. Pre postavljanja transplantata eksperimentalnoj grupi životinja je aplikovana limunska kiselinu na isti na in. Transplantatom je prekrivena ogoljena površina, fiksacija je ura ena pojedina nim šavovima, a rana zašti ena hirurškim pakovanjem. Konrolna grupa životinja je tretirana na isti na in bez limunske kiseline. Životinje su žrtvovane osam nedelja nakon primene odgovaraju ih mukogingivalnih hirurških zahvata. Uzeti materijal je obra en rutinskim postupkom koji je predvi- en za histološka ispitivanja tkiva svetlosnom mikroskopijom. Posle fiksacije u 10% formalinu, materijal je stajao u dekalcinatu 7-10 dana. Parafinski kalupljien materijal je se en na preparate debljine oko 5 m koji su nakon fiksacije na mikroskopskim plo icama bojeni hemotoksin eozinom (HE), Masson- Trihrom -om i Paf- Hallmi-em. Stanje parodontalnih tkiva eksperimentalnih životinja analizirano je svetlosnom mikroskopijom. position. In the control group, the animals were not treated with citric acid. The exposed surfaces on the right side were covered with FGG, i.e. half width flap. The recipient site was prepared by tissue excision and the tooth surface was prepared in the same manner. The graft was taken from the edentulous region lateral to the treated teeth. Prior to FGG positioning in the experimental group, citric acid was applied in the same manner as in animals treated with LPF. The graft covered the exposed root surface, the fixation was done using single sutures and the wound was covered with surgical dressing. The control group was treated without citric acid. The animals were sacrificed eight weeks after the treatment. The material was prepared in a routine manner for histological sampling under light microscopy. After 10% formalin fixation, the material was decalcified for 7-10 days. Paraffin molds were cut in 5 m thick slices which were stained with haematoxylin eosin (HE), Masson-Trichrome and Paf-Hallmi. Periodontal tissue status in experimental animals was analyzed under the light microscope. Rezultati Dobijeni rezultati prikazani su na slikama Na patohistološkim preparatima koji su ura eni osam nedelja posle primene režnja pune debljine utvr eno je slede e. Epitel je sa papilomatozom, a prema pripoju i u sulkusu je atrofi an. U subepitelnom tkivu je prisutna snažna inflamacija iji intenzitet raste prema dubini (sl.1). U elijskom sastavu infiltrata dominiraju monojedarne elije, limfociti, plazmociti i eozinifili. Histološki daju sliku pseudonodula. Vezivna vlakna su dobro formirana. Od dna gingivalnog sulkusa su postavljena pod pravim uglom u odnosu na površinu zuba (sl.2 i 3 ). Na površini korena zuba i duž cementa su prisutni brojni osteoblasti postavljeni gusto u nizu (sl. 4 i 5 ). U krznu gingive se vide snažno dilatirani krvni sudovi (sl.6 ). Vezivno tkivni pripoj je dobar. U pripojnom tkivu su mestimi no prisutna inflamatorna žarišta (sl. 2 ). Pažljivim posmatranjem histopatološkog materijala nisu evidentirani odontoblasti eventualno ošte eni zbog upotrebe limunske kiseline (sl.7). Samo na jednom preparatu se može videti nekroti no žariste pulpe, ali verovatno kao posledica karijesa ili nekog drugog uzroka. Results The results are presented in Figures Patho-histological samples prepared 8 weeks after full width flap showed the following: Epithelium was characterized with papilomatosis and atrophy in areas towards the attachment and sulcus. In the subepithelial tissue, there was a severe inflammatory reaction with an increasing intensity towards deeper parts (Fig. 1). In the cellular composition dominated mononuclear cells, lymphocytes, plasmocytes and eosinophyles. Histologically, they formed a pseudo-nodular image. Connective tissue fibers were well formed. From the bottom of the gingival sulcus, they were perpendicular to the tooth surface (figs. 2 and 3.). On the tooth surface and along cementum, there were numerous osteoblasts in a thick row (figs. 4 and 5). Extremely dilated blood vessels were noticed in the gingival tissue (fig. 6). Connective tissue attachment was satisfactory with occasional inflammatory foci (fig. 2). After careful observation of patho-histological material, no odontoblasts damaged by citric acid were seen (fig. 7). On a single specimen, necrotic pulpal focus was seen, probably caused by caries or other factors.

6 216 Stom Glas S, vol. 54, Slika 1. Mukoperiostalni LPR ra en u kombinaciji sa limunskom kiselinom inflamatorna zona na dnu gingivalnog sulkusa Figure 1. Mucoperiosteal LPF performed in combination with citric acid inflammatory zone at the bottom of the gingival sulcus. Slika 2. Mukoperiostalni LPR ra en u kombinaciji sa limunskom kiselinom pripoj i orijentacija kolagenih vlakana gingive za cement Figure 2. Mucoperiosteal LPF performed in combination with citric acid attachment and orientation of collagen fibers towards cementum. Slika 3. Mukoperiostalni LPR ra en u kombinaciji sa limunskom koselinom pripoj i orijentacija kolagenih vlakana gingive za cement Figure 3. Mucoperiosteal LPF performed in combination with citric acid - attachment and orientation of collagen fibers towards cementum. Slika 4. LPR ra en u kombinaciji sa limunskom kiselinom izgled cemeta posle operacije Figure 4. LPF performed in combination with citric acid cementum, postoperatively.

7 Serbian Dental J, 2007, Slika 5. LPR ra en u kombinaciji sa limunskom kiselinom izgled cementa tretiranih zuba Figure 5. LPF performed in combination with citric acid cementum of the treated teeth. Slika 6. LPR ra en u kombinaciji sa limunskom kiselinom izgled gingive posle operacije Figure 6. LPF performed in combination with citric acid gingiva, postoperatively. Slika 7. LPR ra en u kombinaciji sa limunskom kiselinom izgled pulpe posle operacije Figure 7. LPF performed in combination with citric acid dental pulp, postoperatively. Rezultati histopatoloških ispitivanja parodoncijuma eksperimentalnih životinja posle primene režnja poludebljine su pokazali epitel jasno pripojen za površinu zuba. Prisutan je plitak gingivalni sulkus, blago izražena papilomatoza epitela i mestimi no prisutne erozije na površini. Pripoj vezivnog tkiva uz koren je dobar. Vezivna vlakna grupisana u snopove su postavljena me usobno paralelno, ali koso odnosno tangencionalno u odnosu na površinu zuba. Mestimi no je položaj vlakana i haoti an (sl. 8). Patho-histological results on experimental animals treated with half width flap revealed a clearly distinctive epithelium attached to the tooth surface. A shallow gingival sulcus was present, mild epithelial papilomatosis and occasional erosions on the surface. Connective tissue attachment was satisfactory with parallel fibers grouped in bundles and positioned oblique or tangential in relation to the tooth surface. Occasionally, the orientation of fibers was chaotic (fig. 8).

8 218 Stom Glas S, vol. 54, Slika 8. Mukozni režanj ra en u kombinaciji sa limunskom kiselinom pripoj i orijentacija kolagenih vlakana gingive za cement Figure 8. Mucous flap in combination with citric acid attachment and orientation of collagen fiber towards cementum. Slika 9. Mukozni režanj ra en u kombinaciji sa limunskom kiselinom pseudonoduli u krznu gingive Figure 9. Mucous flap in combination with citric acid pseudo-nodules in the gingival tissue. Slika 10. Mukozni režanj raden u kombinaciji sa limunskom kiselinom izgled cementa posle operacije Figure 10. Mucous flap in combination with citric acid cementum, postoperatively. Slika 11. Mukozni režanj ra en u kombinaciji sa limunskom kiselinom izgled cementa posle operacije Figure 11. Mucous flap in combination with citric acid - cementum, postoperatively.

9 Serbian Dental J, 2007, U subepitelnom tkivu su prisutna sitna žarista inflamacije u iji sastav ulaze monojedarne elije, limfociti i plazmociti. Ova inflamatorna žarista histološki odgovaraju pseudonodulima (sl. 9 ). Prisustvo žarista inflamacije ukazuje na imunološka zbivanja u parodoncijumu. Krvni sudovi su dilatirani, ali manje brojni nego u krznu režnja pune debljine. Ispod vezivno tkivnog pripoja vidi se cement neravnomerne debljine sa znacima proliferacije i poja ane cementogeneze (sl. 10 i11 ). Ovo je naro ito izraženo na apikalnom delu zuba. Tako je u apikalnim partijama cement skoro dva puta deblji nego u koronarnim. Zadebljali cement je hiperplasti an, pravilno slojevite gra e. Paralelni snimak cementa radne i kontrolne strane ukazuje na stimulativno dejstvo limunske kiseline na cementoblaste kojih je broj ano više nego na kontrolnoj strani. Ni na ovim snimcima nisu registrovani znaci oste enja odontoblasta ili vezivnog tkiva pulpe (sl. 12). In the sub-epithelial tissue, there were small inflammatory foci containing mononuclear cells, lymphocytes and plasmocytes. Histologically, these foci corresponded to pseudo-nodules (fig. 9). The presence of inflammtory foci implied there were immunological responses in the periodontal tissue. Blood vessels were dilated but less numerous that in full width flap. Underneath the connective tissue attachment, there was the cementum of uneven width and with signs of proliferation and enhanced cementogenesis (figs. 10 and 11). This was particularly evident in the apical part of the tooth. In these apical parts, cementum was almost twice the thickness of that in coronal parts. Cementum was also hyperplastic with regular laminar structure. A parallel image of cementum on the experimental and control sides revealed a stimulatory effect of citric acid on cementoblasts, whose number increased on the experimental that control side. Furthermore, no damage of odontoblasts or pulpal connective tissue was recorded on these images (fig. 12). Slika 12. Mukozni režanj ra en u kombinaciji sa limuskom kiselinom izgled pulpe tretiranih zuba Figure 12. Mucous flap in combination with citric acid dental pulp of the treated teeth. Diskusija Na izbor eksperimentalnih životinja su uticala ranija pozitivna iskustva koja se prvenstveno odnose na anatomo-histološku gra u parodoncijuma sli nu ljudskim, kao i mogu nost izbora životinja iz istog legla, njihovu zaštitu i kontrolu. Rezultati istraživanja su pokazali da se osam nedelja posle prekrivanja denudiranih površina zuba tretiranih limunskom kiselinom lateralno pomerenim režnjem i slobodnim transplantatom gingive reate men tkiva sastoji od epitela, vezivnog tkiva i novoformiranog cementa. Epitel u obe grupe ekspermentalnih životinja ima karakteristike epitela zdrave gingive. Vezivno tkivni pripoj se sastoji od kolagenih vlakana paralelno grupisanih u snopove. U životinja tretiranih režnjem pune debljine kolagena vlakna su postavljena upravno na površinu zuba. Ispitivani period od osam nedelja je bio dovoljan da novoformirana Discussion The choice of experimental animals was influenced by previous positive experience primarily with regards to anatomo-histological structure of the periodontal tissue, which is similar to human, as well as the possibility to choose animals from the same breed, their safety and control. The present results showed that 8 weeks after covering the exposed tooth surfaces treated with citric acid, laterally positioned flap and free gingival graft, the reattachment consisted of epithelium, connective tissue and newly developed cementum. Epithelium in both experimental groups showed signs of healthy gingival epithelium. The connective tissue attachment consisted of parallel collagen fibers grouped in bundles. In animals treated with full width flap, collagen fibers were located perpen-

10 220 Stom Glas S, vol. 54, vlakna zauzmu funkcionalnu orijentaciju. U životinja tretiranih mukoznim režnjem novoformirana vlakna su kosa i tangencionalna u odnosu na površinu zuba, a mestimi no i haoti no postavljena. Na površini korena duž cementa u obe grupe životinja se mogu videti osteoblasti postavljeni gusto i u nizu. Evidentno je prisustvo novog cementa koji je hiperplasti an i pravilno slojevite gra e. Uo ava se proliferacija i poja ana cementogeneza na modelima životinja tretiranih režnjem pune debljine uz prethodnu aplikaciju limunske kiseline. Paralelni snimak cementa radne i kontrolne strane ukazuje na stimulativno dejstvo kiseline na cementogenezu. Uo ava se i ve e prisustvo krvnih sudova posle primene režnja pune debljine što je posledica od po etka bolje vaskularizacije peteljkastog režnja u odnosu na slobodni transplantat gingive. Chandra i sar. 4 ispituju SEM u in vitro uslovima efekat kondicioniranja na reparatorne sposobnosti parodoncijuma pomo u V79 elija i fibroblasta humanih periodontalnih ligamenata (HPDL). Adekvatno pripremljene površine korena kondicioniraju limunskom kiselinom ph 1,2, EDTA i 10% rastvorom tetraciklin hidrohlorida i destilovanom vodom kao kontrolonom grupom. Eksperiment je ura en na 80 zuba. SEM-om su utvrdili identi no ponašanje V79 elija i HPDL elija na kondicioniranim površinama zuba i to zdravu morfologiju elija na površinama demineralizovanim limunskom kiselinom i EDTA a nepravilan izgled na površinama tretiranim tetraciklinom hidrohloridom i destilovanom vodom. Rezultati eksperimenta ukazuju na povoljan efekat limunske kiseline i EDTA na migraciju, pripoj i morfologiju fibroblasta kao zna ajnog faktora u regeneraciji parodoncijuma. Identi ne rezultate prethodnim dobija i Baby 5. On ogoljene površine parodontopati nih zuba prethodno tretirane razli itim ru nim instrumentima u in vitro uslovima kondicionira limunskom liselinom ( ph 1 ), tetraciklin hidrohloridom (ph 1.8 ), i 8% rastvorom EDTA (ph 7.3) u trajanju od 3 min. SEM je registrovao zna ajno ve i broj fibroblasta pripojen na površinama koje su prethodno kondicionirane bez obzira na vrstu demineralizacije u odnosu na površine samo obradene ru nim instrumentima. Ovo sugeriše na injenicu da je bolji tkivni pripoj na demineralizovanoj površini zuba zbog detoksifikacije površine korena kojom se eliminišu supstance koje deluju inhibitorno na produkciju fibroblasta. Ispituju i reparatorni potencijal kondicioniranja u terapiji parodontopatije Baker i sar. 6 skening elektronskom mikroskopijom dokazuju prisustvo tanke mreže fibrina direktno priljubljene za kondicioniranu površinu dentina uklještenu izmedu elijskih elemenata uglavnom eritrocita. Na nekondicioniranim površinama kontrolne grupe zuba mogu se videti nepravilno rasute amorfne plaže absorbovanih fibroznih proteina i eritrociti mestimi no priljubljeni za površinu dentina. Polaze i od injenice da je o uvanost strukture dentina i biohemijskih karakteristika njegovog matriksa krucijalna u procesu regeneracije parodoncijuma Ruggeri i sar. 7 dicular to the tooth surface. The observation period of 8 weeks was long enough for newly formed fibers to gain functional orientation. In animals treated with the mucous flap, newly formed fibers were oblique and tangential to the tooth surface and occasionally chaotic. Along the cementum in both groups of animals, thick and regularly positioned osteoblasts could be seen. Newly formed hyperplastic, regularly laminar cementum was observed. Proliferation and enhanced cementogenesis were observed in animals treated with full width flap with the application of citric acid. A parallel image of cementum on the experimental and control sides revealed a stimulatory effect of citric acid on cementogenesis. Increased presence of blood vessels was observed in the full width flap group, caused by better vascularization of the shank-like flap compared to free gingival graft. Chandra et al. 4 conducted a SEM study on in vitro effects of conditioning on reparative ability of periodontal tissue with V79 cells and fibroblasts of human periodontal ligaments (HPDL). They conditioned prepared root surfaces with citric acid, ph 1.24, EDTA and 10% solution of tetracycline hydrochloride and distilled water as control. The experiment was done on 80 teeth. SEM revealed the same behavior of V79 and HPDL cells on conditioned root surfaces, healthy cell morphology on surfaces demineralized by citric acid and EDTA and irregular appearance on surfaces treated with tetracycline hydrochloride and distilled water. The results pointed out the favorable effect of citric acid and EDTA on the migration, attachment and morphology of fibroblasts as an important factor in periodontal regeneration. Identical results were reported by Baby 5 who conditioned the exposed surfaces of periodontopathic teeth previously treated with hand instruments in vitro with citric acid (ph=1), tetracycline hydrochloride (ph=1.8) and 8% EDTA (ph=7.3) for 3 min. SEM revealed significantly greater number of fibroblasts attached to previously conditioned surfaces regardless of the source of demineralization compared to surfaces treated with hand instruments only. This suggested that better tissue attachment on the demineralized tooth surface occured due to surface detoxication which eliminated inhibitory substances for fibroblast production. Investigating the reparative potential of conditioning in the treatment of periodontal disease, Baker et al. 6 confirmed, using SEM, the presence of a fine fibrin network directly attached to the conditioned dentin surface clenched between cellular elements, mostly erythrocytes. Irregular amorphous areas of absorbed fibrous proteins and erythrocytes occasionally attached to dentin surface were seen on untreated control surfaces.

11 Serbian Dental J, 2007, Based on the fact that preserving dentinal structure and biochemical characteristics of its matrix were crucial in periodontal regeneration, Ruggeri et al. 7 investigated biochemical characteristics of collagen and chondroitin sulphate in the dentin surface of teeth with periodontal disease after conditioning. The results of their immunohistochemical SEM study showed intensive networks of collagen fibrils and proteoglycanes on tooth surfaces treated with 10% citric acid. Similar was the structure of dentin conditioned with 17% EDTA. On surfaces treated with hand and ultrasonic instruments, the dentinal smear layer was observed with no fibrin network. A SEM study by Vanheusden et al. 8 showed that dentin surface conditioning stimulated the kinetics and morphology of human gingival keratocytes, important in the repeated formation of the attached epithelium. Zaman et al. 9 investigated the migration, attachment and position of cells of human periodontal fibers on periodontopathic cementum and dentin after demineralization with citric acid, EDTA and tetracycline hydrochloride. Their results showed significantly better positioning, migration and orientation of cells in human periodontal fibers on demineralized cementum and dentin compared to the control group. The values of observed parameters did not differ in demineralized cementum compared to dentin. Quantitative analysis revealed no significant difference in the observed parameters in citric acid, EDTA or tetracycline hydrochloride treated teeth. Their results implied that demineralization of periodontopathic teeth was not necessary for the removal of necrotic cementum. Yukna 10 covered the exposed tooth surfaces, previously conditioned with citric acid, with full width flap in combination with enamel matrix derivatives (EMD). Their histological results showed the presence of newly formed periodontal tissue (new cementum, bone and periodontal fibers) in three cases, new connective tissue attachment in another three cases and long attached epithelium in four cases. There were no signs of root resorption, ankylosis or inflammation one month after surgery. Daly 11 investigated the antibacterial effect of citric acid on the dental plaque in vitro. His results revealed a reduced number of aerobic (55%) and anaerobic (30%) microorganisms in the dental plaque on teeth with periodontal disease as early as 24 h after the application of citric acid. Investigating the agar culture of the same teeth, he confirmed that citric acid reduced aerobic and anaerobic microorganism growth by 95% and 80%, respectively. Cochran et al. 12 followed periodontal tissue regeneration in artificial shallow (1 mm and 2 mm) and deep (4 mm and 6 mm) bony defects which were covered with full width flap in combination with extracellular enamel matrix proteins and pre-conditioning of exposed surfaces with EDTA (experimental group) compared to the full width flap coverage and pre-conditioning (control group). Their histological results confirmed newly developed colispituju biohemijske karakteristike kolagena i hondroitin sulfata površine dentina parodontopati nih zuba nakon kondicioniranja. Rezultati njihovog imunohistohemiskog SEM istraživanja su pokazali da se na površinama zuba kondicioniranih 10% limunskom liselinom može uo iti intenzivna mreža kolagenih fibrila i proteoglikansa. Sli na je i struktura dentina kondicioniranog sa 17% EDTA. Na površinama zuba obra enih samo ru nim i ultrazvu nim instrumentima može se videti samo razmazni sloj dentina bez fibrinske mrežice. SEM ispitivanja Vanheusden i sar. 8 su pokazala da se kondicioniranjem površine dentina stimuliše kinetika i morfologija humanih gingivalnih keratocita koji su zna ajni u reformiranju pripojnog epitela. Zaman i sar. 9 ispituju migraciju, pripoj i položaj humanih elija periodontalnih vlakana na parodontopati nom cementu i dentinu nakon demineralizacije limunskom kiselinom, EDTA i tetraciklin hidrohloridom. Rezultati njihove studije su pokazali statisti ki zna ajno bolji položaj, migraciju i orijentaciju elija periodontalnih vlakana na demineralizovanom cementu i dentinu u odnosu na kontrolnu grupu ne-demineralizovanih zuba. Vrednost ispitivanih parametara se nije razlikovala na demineralizovanom cementu u odnosu na dentin. Kvantitativnom analizom nije utvr ena zna ajna razlika ispitivanih parametara u zavisnosti od toga da li su zubi tretirani limunskom kiselinom, EDTA ili tetraciklin hidrohloridom. Rezultati njihovog istraživanja navode i na zakljuc ak da demineralizacijom parodontopati nih zuba nije neophodno ukloniti nekroti an cement. Yukna 10 limunskom kiselinom kondicionira ogoljene površine zuba koje zatim prekriva režnjem pune debljine u kombinaciji sa derivatima glednog matriksa (EMD). Rezultati njegovog histološkog ispitivanja su pokazali prisustvo novog parodontalnog tkiva (novog cementa, kosti i periodontalnih vlakana) u 3 slu aja, novog vezivno tkivnog pripoja tako e u 3 slu aja i prisustvo dugog pripojnog epitela u 4 slu aja. Nije bilo znakova resorpcije korena, ankiloze ni prisutne inflamacije mesec dana nakon operacije. Daly 11 ispituje antibakterijski efekat limunske kiseline na dentalni plak u in vitro uslovima. Rezultati ove studije su pokazali da je ve 24h nakon primene limunske kiseline redukovana koli ina aerobnih (55%) i anaerobnih (30%) mikroorganizama u dentalnom plaku na ekstrahiranim parodontopati nim zubima. Ispitivanjem kulture agara istih zuba ustanovljeno je da limunska kiselina redukuje rast aerobnih i anaerobnih mikroorganizama za 95%, odnosno 80%. Cochran i sar. 12 prate regeneraciju parodoncijuma arteficijelno izazvanih plitkih (1 i 2 mm) i dubokih (4 i 6 mm) koštanih defekata koje prekrivaju režnjem pune debljine u kombinaciji sa ekstracelularnim belan evinama gle nog matriksa pri emu ogoljene površine kondicioniraju neutralnim rastvorom EDTA (eksperimentalna grupa) i režnjem pune debljine tako e uz prethodno kondicioni-

12 222 Stom Glas S, vol. 54, ranje (kontrolna grupa). Rezultati njihovog histološkog istraživanja su pokazali u svim slu ajevima novoformirana kolagena vlakna, cement i kost pet meseci nakon operacije. Širina novoformiranog vezivnog tkiva položaj i orijentacija kolagenih vlakana su u obe grupe životinja nezavisno od dubine defekata bili identi ni. U eksperimentalnoj grupi životinja koronarno od arteficijalnih defekata je registrovana burna reakcija novoformiranog tkiva u smislu velike koli ine novog cementa, kosti i vezivnog tkiva u ve ini slu ajeva. Koštani defekti širine 4-6 mm su bili popunjeni novoformiranim koštanim tkivom. Novoformirana periodontalna vlakna su nezavisno od dubine koštanih defekata bila identi no jednaka. Širina novoformiranog cementa je bila neznatno ve a na zubima sa ve im koštanim defektima. U eksperimentalnoj grupi sa pli im defektima (1 i 2 mm) je registrovano 45% više novoformiranog cementa i 31% više novoformirane kosti u odnosu na kontrolnu grupu. Na životinjama sa prisutnim dubljim koštanim defektima (4 i 6 mm) nije registrovana zna ajna razlika novoformiranog cementnog i koštanog tkiva izme- u eksperimentalne i kontrolne grupe životinja. Kondicioniranje ogoljenih površina parodontopati nih zuba limunskom kiselinom i EDTA ima i široku klini ku primenu u terapiji parodontopatije. Hou i sar. 13 ispituju i efikasnost resorptivnih membrana Atrisorb i Resolut XT na zbrinjavanje koštanih defekata parodontopatije primenjuju kondicioniranje kiselinom. Rezultati njihove studije su zadovoljavaju i u smislu smanjenja nivoa pripojnog epitela, dubine parodontalnih džepova, i vrednosti gingivalnog i plak indeksa u postoperativnom periodu. Rupprecht i sar. 14 takode koriste limunsku kiselinu za kondicioniranje korenova zube koje hirurški tretiraju koronarno pomerenom režnjem (KPR) u kombinaciji sa kalcijum hidroksilapatitnim cementom (HAC) i dobijaju zna ajno bolje rezultate u odnosu na grupu bolesnika tretiranih samo klasi nim KPR. Sli ni su rezultati klini kog rada Cheng 15, Decima 16 Bouchard 17 i drugih. lagen fibers, cementum and bone 5 months after treatment in all cases. The width of the newly formed connective tissue attachment and the orientation of collagen fibers in both groups, irrespective of defect depth, were identical. In most cases in the experimental group, a severe reaction of the newly formed tissue with new cementum, bone and connective tissue was observed coronally from the artificial defects. Bony defects 4-6 mm wide were filled with newly developed bone. Newly formed periodontal fibers were identical, irrespective of the depth of bony defects. The width of the new cementum was slightly greater in teeth with larger bony defects. In the experimental group with shallower defects (1 mm and 2 mm), 45% more newly formed cementum and 31% more newly formed bone were recorded compared to the control group. In animals with deeper bony defects (4 mm and 6 mm), no significant difference in the amount of newly formed cementum and bone was recorded between the experimental and control groups. Conditioning of exposed root surfaces of teeth with periodontal disease with citric acid and EDTA has wide clinical application in the treatment of periodontal disease. Hou et al. 13 applied acid conditioning in a study of efficiency of resorptive membranes, Atrisorb and resolute XT, in treating bony defects. Their results were satisfactory in terms of reduction of gingival attachment, depth of periodontal pockets and gingival and plaque index values postoperatively. Rupprecht et al. 14 also used citric acid for root surface conditioning during coronally positioned flap (CPF) treatment in combination with calcium hydroxyapatite cement (HAC) and reported significantly better results compared to the control group, treated with only classical CPF. Similar results were reported by Cheng 15, Decim 16, Bouchard 17 and others. Literatura / References 1. Stevanovic R. Primena rezanj operacije u otklanjanju funkcionalnih i estetskih nedostataka parodoncijuma. Magistarski rad, Stomatoloski Fakultet, Beograd Urist MR. Bone histogenesis and morphogenesis in implants of demineralized enamel and dentin.oral Surg 1971; 29: Register AA, Burdick F. Accelerated reattechment with cementogenesis to dentin, demineralized in situ. J Periodont 1975; 46: Chandra RV, Jagotia GC, Bhat KM. The attachment of V79 and human periodontal ligament fibroblasts on periodontally involved root surfaces following treatment with EDTA, citric acid, or tetracycline HCL: An SEM in vitro study. Contemp Dent Pract 2006; 7: Babay N. Attachment of Human Gingival Fibroblasts to Periodontally Involved Root Surface Following Scaling and/or Etching Procedures: A Scanning Electron Microscopy Study. Braz Dent J. 2001; 1: Baker PJ, Rotch HA, Trombelli L, Wikesjo UME. An in vitro screening model to evaluate root conditioning protocols for periodontal regenerative procedures. J Periodont. 2000; 71: Ruggeri Jr A, Prati C, Mazzoni A, Nucci C, Di Lenarda R., Mazzotti G, Breschi L. Effects of citric acid and EDTA conditioning on exposed root dentin: An immunohistochemical analysis of collagen and proteoglycans. Archiv Oral Biol 2007; 52: Vanheusden AJ, Goffinet G, Zahedi S, Nusgens B, Lapie`re CM, Rompen EH. In vitro stimulation of human gingival epithelial cell attachment to dentin by surface conditioning. J Periodont 1999; 70: Zaman KU, Sugaya T, Hongo O, Kato H. A study of attached and oriented human periodontal ligament cells to periodontally diseased cementum and dentin after demineralizing with neutral and low ph etching solution. J Periodont 2000; 71:

13 Serbian Dental J, 2007, Yukna R, Mellonig JT. Histologic evaluation of periodontal healing in humans following regenerative therapy with enamel matrix derivative. A 10-case series. J Periodont 2000; 71: Daly CG. Anti-bacterial effect of citric acid treatment of periodontally diseased root surfaces in vitro. J Clin Periodont 1982; 9: Cochran DL, King GN, Schoolfield J, Velasquez-Plata D, Mellonig JT, Jones A. The effect of enamel matrix proteins on periodontal regeneration as determined by histological analyses. J Periodont 2003 ;74: Hou, LT, Yan, JJ, Tsai AY, Lao CS, Lin SJ, Liu CM. Polymer-assisted regeneration therapy with Atrisorb barriers in human periodontal intrabony defects. Journal of Clinical Periodontology Volume 31, Issue 1, January 2004, Pages Rupprecht RD, Horning GM, Towle HJ. A clinical evaluation of hydroxyapatite cement in the treatment of Class III furcation defects. J Periodont 2001;72: Cheng YF, Chen JW, Lin SJ, Lu HK. Is coronally positioned flap procedure adjunct with enamel matrix derivative or root conditioning a relevant predictor for achieving root ceverage? A systemic review. J Periodont Res. 2007;42: Decima NRM, Mendes ABN Jr, Arthur BN. Root Coverage of Large Localized Gingival Recession: A Biometric Study Braz Dent J.1997; 8: Bouchard P, Nilv eus R, Etienne D. Clinical evaluation of tetracycline HCl conditioning in the treatment of gingival recessions. A comparative study. J Periodont 1997; 68: Autor odgovoran za korespondenciju Ružica Nedeljkovi Klinika za Bolesti zuba Stomatološki fakultet Beograd Rankeova Beograd Address for correspondence Ružica Nedeljkovi Dept. of Restorative Dentistry and Endodontics School of Dentistry, Belgrade Rankeova Belgrade, Serbia