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1 Serbian Dental J, 2006, UDC: : ]-076 ISSN COBISS. SR-ID Eksperimentalna skening elektronsko mikroskopska izučavanja patološke deninogeneze u posteruptivnoj fazi zuba Experimental scanning electron microscopic investigation of pathological dentinogenesis in posteruptive tooth phase DOI: /SGS V Jovan Vojinovi 1, Radoje Stevanovi 2, Slobodanka Hrva anin 3 1 Medicinski fakultet Banja Luka, Odsjek za stomatologiju, Katedra za de ju i preventivnu stomatologiju 2 Stomatološki fakultet u Beogradu, Klinika za de ju i preventivnu stomatologiju 3 Medicinski fakultet Banja Luka, Odsjek za stomatologiju, Katedra za oralnu higijenu ORIGINALNI RAD (OR) ORIGINAL ARTICLE KRATAK SADRŽAJ U radu su prikazani rezultati Skening elektronsko mikroskopskih ispitivanja patološke dentinogeneze, na humanim zubima i zubima pasa, u razli itim formativnim fazama i pod razli itim patološkim okolnostima. Konstatovano je da se patološka dentinogeneza odvija na genetski predodre en na in bez obzira na vrstu iritacije. Struktura patološkog dentina zavisi od ja ine iritacije i dužine njenog dejstva. Iritacije slabijeg intenziteta uslovljavaju lu enje tubularnog dentina sa nepravilnim pružanjem dentinskih tubula. Stepen nepravilnosti zavisio je od ja ine iritacije. Jake iritacije ošte uju ili uništavaju odontoblaste, a na njihovo mesto se lociraju novodiferencirane elije. Dentin koji one lu e je po strukturi utoliko nepravilniji, ukoliko je stepen njihove diferencijacije niži. Novodiferencirane elije odmah zapo inju sa lu enjem, u po etku atubularnog, a kasnije dentina u kome se postepeno stvaraju tubuli. Odontoblasti su izuzetno otporne elije, tako da i pod jakim iritacijama neku od njih mogu da sa uvaju integritet i nastave da formiraju pojedina ne tubule, koji se vide unutar amorfne dentinske mase. Otpornost odonoblasta na dejstvo iritacija je ograni eno, naro ito kada one dugo deluju i na taj na in iscrpljuju formativni potencijal ovih elija. Ovo ima za posledicu lu enje dentina sve nepravilnije strukture, da bi u završnoj fazi elijskog života izlu eni dentin predstavljao bezstrukturnu amorfnu masu. Ove biološke injenice se moraju uzeti u obzir prilikom odre ivanja terapije pulpodentinskog kompleksa. Terapija treba da bude tako koncipirana, da obezbedi nesmetani oporavak ošte enih odnontoblasta, odnosno diferencijaciju novih formativnih elija koje su zamenile uništene odontoblaste. Drugim re ima, poštuju i biološke principe treba omogu iti elijama da neometano obave svoj genetski predodre eni zadatak u odbrambeno-reparativnom procesu. Ključne reči: Skening elektronska mikroskopija, patološki dentin SUMMARY In the article are presented results of SEM study of pathological dentinogenesis on the human and dogs tooth in the different formative stages. The pathological dentinogenesis take place in the genetically predetermined way no matter of type of an irritation. The structure of pathological dentin depends upon the level of irritation and its duration. Lower level irritation produce the secretion of tubular dentine with irregular tubular direction, higher irritation stronger irregularities. Strong irritations damage odonoblastes, and they are replaced by undifferentiated cells. Lower level of differentiation produce more irregular dentine. New cells momentarily starts secretion, at the beginning the atubular dentine, and latter tubules arrear. The odontoblasts are very resistant cells, so even under of very strong irritants some of them could survive and continue formattiing individual tubules detected within amorphous dentinal tissue. The resistance of odontoblasts is limited, specially when irrritations are long enough exhausting formative potential of cells. Excreted dentinal tissue becomes more and more irregular. These biological facts should be consider in teh therapy of puplodentinal complex. Therapy have to preserve conditions for undisturbed recovery of damaged odontoblasts, or new undifferentiated cells. In the other words biological principles insist on the preservation of conditiions for the normal expression of formative cells genetically tasks. Key words: SEM, pathological dentine

2 96 Stom Glas S, vol. 53, Patološka dentinogeneza je oblast koja još uvek nije u potpunosti definisana. Mnogi problemi od nau nog, ali i od klini kog zna aja, ostali su nerazjašnjeni. Veliki broj autora je pokušavao da objasni suštinu lu enja dentina pod patološkim okolnostima. Brojne publikacije iz ranijih perioda kao i neka savremenih istraživanja objašnjavaju ovaj problem genetski uslovljenim mehanizmima. (1) Mišljenja razli itih autora o navedenom problemu su razli ita, pa esto i suprostavljena. Istraživanja u ovoj oblasti su zato mnogobrojna kao i koriš ene metode. Ipak u izu avanju strukture dentina naj- eš e se koriste svetlosno mikroskopska istraživanja i skening elektronsko mikroskopska istraživanja, a za izu avanje sastava dentina sodijum dodecil sulfat poliakrilamidna elektroforeza SDS PAGE, i dvodimenzionalna elektroforeza 2D, pružaju veliku pomo u razjašnjenju injenica vezanih za patološku dentinogenezu. (2) Rezultati obimnih eksperimentalnih svetlosno mikroskopskih izu avanja prikazani su ranije (3), a u ovom radu je ovaj problem analiziran u svetlu skening elektronsko mikroskopskih izu avanja (SEM), patološke dentinogeneze. Osnovni cilj rada je bio da se SEM izu avanjima dopune, potvrde ili opovrgnu nalazi dobijeni metodom svetlosne mikroskopije (SM). S tim u vezi cilj ovog eksperimentalnog istraživanja bio je da se analizira po etna faza lu enja patološkog dentina i odnos prvobitno stvorenog patološkog dentina prema regularno formiranom dentinu, da se analizira promena u strukturi patološkog dentina u toku njegovog sukcesivnog stvaranja, odnosno analizira struktura dentina izlu enog u procesu izumiranja pulpe i struktura patološkog dentina u zavisnonsti od vrste i intenziteta iritacije. Pathological dentinogenesis is an area still not completely defined. Many problems of scientific, but also clinical significance are left unexplained. Many authors have tried to explain the essence of dentin production under pathological circumstances. A lot of publications, from earlier periods but also some contemporary research explain this problem with genetically induced mechanisms (1). Yet, opinions of different researchers are different, very often opposed. Therefore, research in this area is substantial as well as methods used. In investigating dentinal structure, light and scanning microscopy are most commonly used. In investigating dentinal substance, sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and diagonal electrophoresis (2D) are mostly helpful in explaining facts associated with pathological dentinogenesis (2). Results of large experimental light microscopical research were published earlier (3) so in the present study, the problem of pathological dentinogenesis will be analyzed in the light of scanning electron microscopy (SEM). The main aim of the study was to supplement, verify or disprove the results of light microscopical investigation (LM). In addition to this, the aim of this experimental research was to analyze the initial phase of pathological dentin production and the relationship of initially produced pathological dentin with regular dentin, to analyze change in the structure of pathological dentin during its successive production as well as the structure of dentin produced during pulp senescence and the structure of pathological dentin in relation to the type and intensity of irritation. Materija i metod Kao materijal u istraživanjima koriš ena su tri humana i jedan zub psa. Od tri humana zuba dva su bila mle na i jedan stalni. Jedan mle ni zub je bio sa amalgamskim ispunom a jedan stalni i jedan mle ni zub su bili bez ispuna ali sa nele enim karijesom. Na jednom stalnom zubu psa ura ena su dva kaviteta od kojih je jedan ispunjen amalgamom a drugi je bio bez ispuna i sa otvorenom pulpom. Za skening-elektronsko mikroskopsku analizu (SEM) zubi su pripremljeni na slede i na in: (Tab. 1) Na drugom donjem levom stalnom premolaru psa preparisani su kaviteti na okluzalnoj i bukalnoj površini karbidnim borerima uz vodeno hla enje. Broj obrtaja u gle i iznosio je /min, a u dentinu /min. Dubina okluzalnog kaviteta je bila 1,5mm. Bukalni kavitet je preparisan do granice sa pulpom (pulpa se nazire, ali bez krvarenja). Okluzalni kavitet je ispunjen amalgamom, bez postavljanja podloge na dentin, a bukalni kavitet (sa nehemoragi nom trepanacijom) je ostavljen otvoren i izložen dejstvu oralne sredine u toku narednih 15 dana (Sl. 1) Materials and methods As material in this study, 3 human and one dog s teeth were used. From the 3 human teeth, 2 were deciduous and 1 was permanent. One deciduous tooth was amalgam-restored while the other deciduous and the permanent tooth had primary caries, with no restoration. In the dog s tooth, 2 cavities were made: one was restored with amalgam and the other was left without restoration, and with exposed pulp. Teeth were prepared for SEM in the following way: On the second lower permanent dog s molar occlusal and buccal cavities were prepared with carbide burs with water cooling. The speed was 13000rpm in enamel and rpm in dentin and cavity depth was 1.5 mm. Buccal cavity was prepared to the pulp chamber (pulp is exposed, but no hemorrhage). Occlusal cavity was restored with amalgam with no dentinal liner while the buccal cavity was left open and exposed to the oral environment for 15 days (Fig. 1)

3 Serbian Dental J, 2006, Slika 1 SEM fotografija zuba psa: A. Na okluzalnoj površini ispreparisan je plitak kavitet (k), i duboki kavitet sa nehemoragi nom trepanacijom na bukalnoj strani (P). Okluzalni kavitet je bio ispunjen amalgamom a bukalni je ostavljen 15 dana otvoren prema usnoj šupljini; (20x) B. Mesno nehemoragi ne trepanacije na bukalnoj površini i patološki dentin nepravilne strukture (S). Tubularni patološki dentin (t) dalje od mesta trepanacije; (100x) C. Okluzalni kavitet: (o) Niz odontoblasta bez prekida na grani noj liniji sa dentinom; (1200x) D. (S) Mesto nehemoragi ne trepanacije dentina; patološka+ tubularna struktura; (p) Tubularni dentin sa nepravilnim pravcem pružanja kanali a; (150ox) E. Granica (h) izme u regularnog dentina (R) i patološkog dentina (X). Mestimi ni spoj (h) obe vrste dentina: treba zapaziti pojedina ne protoplazmati ne produžetke (p) koji se nastavljaju iz regularnog dentina; (2600x) F. Protoplazmati ni produžetak koji se neprekinuto nastavlja iz regularnog dentina. (860ox) Figure 1. SEM photograph of a dog s tooth: A. A shallow cavity (k) prepared on occlusal surface and deep caity with non-hemorrhage pulp exposure buccally (P). Occlusal cavity was filled with amalgam and the buccal was exposed to the oral cavity for 15 days (20x) B. The site of non-hemorrhage trepanation on the buccal surface and pathological dentin of irregular structure (S). Tubular pathological dentin (t) farther away from the trepanation site. C. Occlusal cavity: (o) Continuous odontoblast layer bordering dentin (1200 x) D. (S) The site of non-hemorrhage trepanation; pathologic and tubular structure ; (p)- tubular dentin with irregular direction of dentinal tubules (1500x) E. Junction (h) between regular (R) and pathological dentin (X). Occasional combination (h) of both types of dentin: notice single protoplasmatic processus (p) out of regular dentin (2600x) F. Protoplasmatic processus continuing from regular dentin without disruption (8600x) Na bukalnoj površini humanog gornjeg desnog mle nog o njaka izva enog iz ortodonstkih razloga, postojao je nele eni karijes na mezijalnoj površini. Zub je u pripremi za SEM prelomljen po sredini karijesnog kaviteta, pri emu je na liniji preloma ostalo tkivo pulpe (Sl. 2) Na humanom stalnom donjem o njaku nele ena karijesna lezija je postojala na obe aproksimalne površine, a krunica zuba je bila skoro destruirana i sa otvorenom pulpnom komorom. U pripremi za SEM zub je prelomljen na dve polovine i dva karijesna kaviteta (Sl. 3) Na okluzalnoj površini humanog donjeg drugog mle nog molara iji su korenovi bili delimi no resorbovani, ispreparisan je kavitet dubine oko 1,5mm i ispunjen je amalgamom, bez prethodnog postavljanja podloge. Posle godinu dana zub je izva en. Resorpcija korenova je u me uvremenu dostigla do 1 mm ispod bifurkacije. U pripremi za SEM zub je prelomljen po sredini kaviteta. Na There was primary caries on the buccal side of human deciduous upper right canine extracted from orthodontic reasons. During preparation for SEM, the tooth was fractured along carious cavity with pulp tissue in the fracture line (Fig. 2) There was primary caries on both proximal sides of human permanent lower canine and the tooth crown was almost destructed and had the exposed pulp chamber. During preparation for SEM, the tooth was fractured in 2 halves and 2 carious cavities (Fig.3) A cavity of 1.5 mm in depth was prepared on occlusal surface of human deciduous lower second molar with partially resorbed roots. The cavity was restored with amalgam with no liner. After a year, the tooth was extracted. Meanwhile, root resorption reached 1 mm below furcation. During preparation for SEM, the tooth was fractured along cavity middle. On the fractured surface, there was

4 98 Stom Glas S, vol. 53, prelomljenoj površini zadržao se samo mali deo pulpe uz zid kanala korena u nivou bifurkacije (Sl. 4) U daljoj pripremi za SEM, svi zubi su uvani u 2,5% rastvoru natrijum hipohlorata u trajanju od 12 asova, zatim su isprani destilovanom vodom i dehidrirani u alkoholu. Nakon toga su stavljeni u vakum komoru i prekriveni slojem zlata debljine oko 30A za SEM analizu. little pulp tissue along root canal in the level of furcation (Fig.4) In the subsequent preparation for SEM, teeth were stored in 2.5% sodium-hypochlorite for 12 hrs, washed with distilled water and dehydrated in alcohol. Afterwards, they were put in vacuum chamber and covered with gold in 30Å depth. Slika 2 SEM fotografija mle nog o njaka sa nele enim karijesom: A. Granica izme u regularnog i patološkog dentina (C), sa mestimi nim prekidom tubula; (K) Kavum pulpe; (R) Regularno formirani dentin; B. Demineralizovani prostori (S) u patološkom dentinu. Treba zapaziti da stuktura patološkog dentina postaje atubularnija prema kavumu pulpe. Figure 2. SEM photograph of deciduous canine with persisting caries: A. Junction between regular and pathological dentin (C) with occasional tubule disruption; (K) pulp chamber; (R) regularly formed dentin B. Demineralized spaces (S) in pathological dentin. Notice that structure of pathological dentin becomes more atubular towards pulp chamber Slika 3 SEM fotografija stalnog o njaka sa nele enim karijesom: A. Razlikuju se: (P) Slojevi regularnog karijesom destruiranog dentina; (K) Skleroziranog dentina; i (m) Patološkog dentina, uz kavum pulpe sa (U) nekroti nim sadržajem; B. Slojevi prikazani na slici A, na ve em uveli anju: (P) Demineralizovani karijesni regularni denitn; (K) Sklerozirani dentin; (M) Patološki iregularni dentin. Dentinski kanali i imaju nepravilniji pravac, pružajui se prema kavumu nekroti ne pulpe (U); (g) granica izme u skleroziranog i patološkog dentina; (h) Granica izme u regularnog destruiranog dentina i skleroziranog dentina; C. Treba zapaziti i oštre ivice deninskih tubula bez protoplazmati nih produžetaka u sklerozirnom dentinu; (K) Sklerozirani dentin; Granica sa regularnim dentinom (R) je uglavnom ravna; D. Ve e uveli anje granice (h) regularnog (R) i skleroziranog dentina (K) Figure 3. SEM photograph of permanent canine with persisting caries: A. There are layers : (P) of dentin regularly destroyed by caries; (K) - of scleroyized dentin and (m) pathological dentin near pulp chamber with (U) necrotic content B. Layers from fig. A in higher magnification: (P) demineralized carious regular dentin; (K) sclerotized dentin; (M) pathological irregular dentin. Dentinal tubules have more irregular direction towards pulp chamber with necrotic pulp (U); (g) junction between sclerotized and pathological dentin; (h) junction between regularly destroyed and sclerotized dentin C. Notice sharp edges of dentinal tubules with no protoplasmatic processus in sclerotized dentin: (K) sclerotized dentin; junction with regular dentin (R) is mostly flat D. Higher magnification of junction (h) between regular (R) and sclerotized dentin (K)

5 Serbian Dental J, 2006, Rezultati Na drugom donjem levom premolaru psa ispod kaviteta dubine oko 1,5mm koji je ispreparisan na okluzalnoj površini i ispunjen amalgamom bez prethodnog postavljanja podloge, nakon 15 dana nije registrovan patološki dentin (Sl. 1A detalj K). Nasuprot ovom kavitetu pulpo dentinska granica je neošte ena sa neprekinutim nizom odontoblasta (Sl. 1C detalj O). Na istom zubu, ali na bukalnoj površini, promene su bile izrazite. Na mestu gde se pulpa nazirala (nehemoragi na trepanacija) (Sl. 1A detalj P), posle 15 dana, uprkos prisustvu bakterija iz usne duplje, zapo elo je formiranje patološkog dentina sa izrazitom tendencijom zaštite najdublje ta ke na dnu kaviteta (Sl. 1B detalj S, 1D detalj S). Ovaj dentin je atubularne strukture. Na mestima koja su udaljena od nehemoragi ne trepanacije, izlu en je patološki dentin tubularne strukture (Sl. 1D detalj P). Na tim mestima, sloj patološkog tubularnog dentina odvojen je od regularnog amorfnom tubularnom masom. Vide se šupljine bez mineralizovane mase (Sl. 1E detalj H). Na mestu ahomoragi ne trepanacije, kroz nemineralizovanu amorfnu masu provla e se pojedina ni tubuli unutar kojih se mogu videti ponegde produžeci odontoblasta (Sl. 1E detalj P). Unutar ovih tubula kao i oko protoplazmati nih produžetaka, treba zapaziti odsustvo peritubularnih vlakana (Sl. 1F). Na humanom mle nom o njaku ispod nele enog karijesa, patološki dentin se jasno razlikuje od regularnog (Sl. 2A detalj C). Na granici koja je jasno nazna ena, postoje mestimi no šupljine, i mestimi no deblji sloj atubularnog dentina, a pojedini dentinski kanali i se bez prekida nastavljaju iz regularnog u patološki dentin (Sl 2A). Broj tubula se smanjuje prema pulpi da bi poslednji sloj bio atubularan. Poslednji izlu eni slojevi atubularnog dentina su interglobularne strukture sa pojedina nim demineralizovanim prostorima (Sl. 2B). O igledno je da nije postojao sloj odontoblasta, a diemineralizacija je bila prime ena i unutar dentinskih kanali a (Sl.. 2B detalj S). Na humanom stalnom o njaku bez pulpnog tkiva, sa karijesnim kavitetom sa mezijalne i distalne strane, sloj patološkog delimi no destruiranog dentina jasno je nazna- en (Sl. 3A detalj P, 3B detalj P). Prvi sloj ispod dna karijesnog kaviteta je skleroziran sa oštrim hipermineralizovanim zidovima suženih tubula (Sl. 3A detalj K, 3B detalj K, 3C detalj K, 3D detalj K). Ispod ove zone nalazi se sloj izrazito iregularnog dentina (Sl. 3A detalj M, 3B detalj M). Njegova granica sa skleroziranim dentinom je neravna (Sl. 3B detalj g, 3C detalj g), dok je granica skleroziranog dentina sa regularnim jasna i ravna (Sl. 3B detalj h, 3C detalj h, 3D detalj h). U iregularnom sloju patološkog dentina tubuli su malobrojni i nepravilnog pravca pružanja, sa nemineralizovanim prostorima haoti no pore anim (Sl. 3A detalj M, 3B detalj M). Mestimi no se mogu videti i dentinski kanali i koji prolaze kroz nemineralizovani sloj. Results There was no production of pathological dentin (Fig.1A, detail K) on the second lower permanent dog s molar, under the occlusal cavity of 1.5 mm deep which was restored with amalgam without the liner(fig.1a, detail K) 15 days after the procedure. Opposite from this cavity, pulp-dentin border is intact with the continuous odontoblast sequence (Fig.1C, detail O). On the same tooth but on the buccal surface, changes were distinctive. At the site of exposed pulp (Fig. 1A, detail P), 15 days after the procedure, in spite of the presence of oral bacteria, production of pathological dentin began with the distinctive tendency to protect the deepest point of the cavity (Fig. 1B, detail S, 1D, detail S). This dentin had atubular structure. Pathological dentin of tubular structure was produced in sites away from the pulp exposure (Fig. 1D, detail P). In these sites, pathological tubular dentin was severed from the regular dentin by amorphous tubular mass. Voids can be seen without mineralized mass (Fig. 1E, detail H) At the site of non-hemorrhage trepanation, single tubules containing odontoblast processus passed through non-mineralized amorphous mass (Fig. 1E, detail P). Within these tubules, as well as around protoplasmatic processus, the absence of peritubular fibres should be noticed (Fig. 1F) In human deciduous canine under primary nonrestored caries, pathological dentin was clearly distinguished from the regular (Fig. 2A, detail C).On a clear demarcation, there were occasionally voids and thicker atubular dentin while some dentinal tubules continuously passed from regular into pathological dentin (Fig. 2A). The number of tubules was lesser towards the pulp and the last layer was completely atubular. Last produced layers of atubular dentin had interglobular structure with few demineralized spaces (Fig. 2B). Clearly, there was no layer of odontoblast cells and demineralization was seen even within dentinal tubules (Fig. 2B, detail S). In human permanent canine without pulp tissue but with carious cavities mesially and distally, a layer of pathological partially destroyed dentin was clearly visible (Fig. 3A, detail P, 3B, detail P). The first layer under the carious cavity was sclerotized with sharp hypermineralized walls of narrowed tubules (Fig. 3A, detail K, 3B, detail K, 3C, detail K, 3D, detail K). Underneath this zone, there was a layer of highly irregular dentin (Fig. 3A, detail M, 3B, detail M). Its demarcation towards sclerotized dentin was uneven (Fig. 3B, detail G, 3C, detail G), while the demarcation between sclerotized and regular dentin was clear and even (Fig. 3B, detail H, 3C, detail H, 3D, detail H). In irregular layer of pathological dentin, there were few tubules with irregular direction and chaotic non-mineralized spaces among them (Fig. 3A, detail M, 3B, detail M). Occasionally, dentin tubules passing through the non-mineralized layer could be seen.

6 100 Stom Glas S, vol. 53, Dentin formiran posle preparacije kaviteta I klase (ne dublji od 1,5 mm ) na humanom mle nom molaru, po strukturi se ne razlikuje od regularnog dentina (Sl. 4). Prema dnu kaviteta svi dentinski kanali i su zatvoreni amorfno atubularnom mineralizovanom masom (Sl. 4C detalj e). Pulpodentinska granica je bila jasno nazna ena sa mestimi no prekinutim odontoblastnim produžecima. Oni su bili vidljivi samo neposredno pored pulpe kako se i o ekivalo (Sl. 4D detalj p). Jasno je bio nazna en i predentinski sloj. Odontoblastni niz je bio neprekinut, uprkos uznapredovaloj resorpciji korenova (Sl. 4D- detalj O). Dentin, produced after class I cavity preparation (not deeper than 1.5 mm) in human primary molar, had the same structure as regular dentin (Fig. 4). All dentinal tubules towards the cavity bottom were closed with amorphous atubular mineralized mass (Fig. 4C, detail E). Pulp-dentnin junction was clearly visible with occasional discontinuity in odontoblast layer. These cells were visible only in the close vicinity of pulp tissue, as expected (Fig. 4D, detail P). Predentin layer was also clearly visible. Odontoblast layer was continuous, in spite of progressive root resorption (Fig. 4D, detail O). In a marked area, there was a sudden change in dentinal tubules direction, in other words, irregular-tubular dentin (Fig. 4A, detail G, 4B, detail G). Slika 4 SEM fotografija humanog mle nog molara na kome je ura ena plitka preparacija kaviteta i postavljen amalgamski ispun. Zub je izva en posle godinu dana. A. Granica izme u regularnog i iregularnog dentina (g) je samo na jednom malom delu dentina sa uzanim slojem patološkog dentina. Na drugim mestima nije bilo patološkog dentina; B. Sloj iregularnog tubularnog dentina (G) detalj sa slike A. Dentinski kanali i brzo dobijaju regualarni pravac pružanja; C. Dentinski kanali i su prema dentinu u kavitetu zatvoreni bez strukturnom amorfnom masom denitna (e); (S) ispuna kaviteta; D. Granica sa pulpom ne pokazuje patološke strukrure: (O) Neprekinut niz odontoblasta; (P) Dentinski kanali i sa protoplazmati nim produžecima. Figure 4. SEM photograph of human deciduous molar with shallow cavity preparation and amalgam restoration. Tooth was extracted after a year. A. junction between regular and irregular dentin (g) only in a small area of dentin with narrow layer of pathological dentin. There was no pathological dentin in other sites. B. A layer of irregular tubular dentin (G) detail from fig. A. Dentinal tubules quickly recover regular direction C. Dentinal tubules towards cavity dentin are closed with amorphous mass of dentin (e); (S) restoration D. Junction towards pulp tissue shows no pathological structures: (O) continuous odontoblast layer; (P) dentinal tubules with protoplasmatic processus.

7 Serbian Dental J, 2006, Diskusija Rezultati SEM istraživanja potvdili su nalaze nedavno objavljenih svetlosno mikroskopskih (SM) istraživanja (3). Potvr eno je da u patološkoj dentinogenezi u estvuju isklju ivo elije odontoblasti. Odontoblasti na iritaciju reaguju individualno. Dešava se da se ak i pod teškim patološkim okolnostima o uvaju pojedini odontoblasti, koji nastavljaju da lu e tubularni dentin (Sl. 1E tdetalj x, p: 1F). Formiranje patološkog dentina obavlja se odmah po dejstvu iritacije i u okolnostima bakterijske infekcije (Sl. 1). Ovaj dentin je najobimniji i najnepravilniji po strukturi upravo na onim mestima na kojima je iritacija bila najja a (Sl. 1D detalj S). Na tim mestima ne postoji regularni odontoblastni niz. U periodu od 15 dana zapažena su brojna nemineralizovana mesta kroz koja se provla e samo pojedina ni o uvani odontoblastni produžeci (Sl. 1E, i 1F). Prema tome, patološki dentin u slu aju dejstva jakih iritacija produkuju novodiferncirani odontoblasti odmah po dejstvu iritacija i pre nego što su dostigli kona ni stepen diferencijacije. Na ovo su ukazali i drugi autori Mestimi ne zone tubularnog dentina u sklopu sa atubularnim, na preparatima dobijenim svetlosnom mikroskopijiom (3), mogu se objasniti prisustvom pojedina nih otpornijih elija koje su o uvale svoj integritet i uprkos jakim iritacijama nastavile sa formativnom aktivnoš u. Ovo je još jasnije potvr eno tokom SEM istraživanja (Sl. 1E, i 1F). Struktura patološkog dentina se menja u toku sukcesivnog stvaranja i u slu aju slabljenja ili prestanka dejstva iritacija, kao i u slu aju postepenog izumiranja pulpe i odontoblasta u njoj. Kod slabih i kratkotrajnih iritacija promene su isklju ivo u pravcu pružanja dentinskih kanali a i to samo na onim mestima na kojima su odontoblasti ošte eni (Sl. 4A detalj g, 4B detalj g). Promene pravca dentinskih kanali a samo na mestima na kojima su pojedini odontoblasti ošte eni još su evidentnije (Sl. 4B detalj g), nego svetlosno mikroskopskim preparatima (3). Ovo je dokaz da odontoblasti reaguju individualno zahvaljuju i individualnom odbrambenom intracelularnom potenicjalu. Interesantno je zapaziti da su na dnu kaviteta posle plitke preparacije otvori dentinskih kanali a obliterirani amorfnom masom sli no atubularnom dentinu u toku patološke dentinogeneze. Ovo ukazuje na mogu uticaj odontoblasntih produžetaka ili dentinske te nosi u tubulima, na šta je ukazao još Foreman (1988). Nalazi autora tako e potvr uju postavku Brannstroma (6,7,8,9) da se odontoblastni produžeci nalaze samo 0,7 mm od pulpe, što ukazuje na njihovu ulogu isklju ivo u formiranju tubula (komparativno Sl. 4C i 4D). U slu aju dugotrajnih iritacija u vidu nele enog karijesa kada je vitalitet pulpe ipak o uvan, patološka dentinogeneza se odvija na isti na in kao i posle mehani kih ili hemijskih iritacija (Sl. 2A). Granica izme u regularnog i patološkog dentina je neravna i na njoj se dentinski kanali i mestimi no Discussion Results of this SEM study confirmed the results of recently published light microscopical investigation (LM) (3). It is confirmed that only odontoblast cells take part in pathological dentinogenesis. Odontoblasts react individually on irritation. Even under severe pathological circumstances some odontoblasts survive and continue to produce dentin (Fig. 1E, detail X, P; 1F) Pathological dentin formation begins immediately after irritation and in bacterial infection (Fig. 1). This dentin is thickest and most irregular in structure in those places where irritation was most severe (Fig. 1D, detail S). In these places, there is no regular odontoblast cell layer. In 15-day-period, numerous non-mineralized areas with few remaining odontoblast processus can be seen (Fig. 1E and 1F). Therefore, pathological dentin in cases of severe irritation is produced by newly differentiated odontoblasts immediately after irritation, even before these cells reach the final degree of differentiation. Other authors states this, too. Occasional zones of tubular with atubular dentin in light microscopy samples (3), can be explained with the presence of a few more resistant cells that maintain their integrity and, despite severe irritation, continue their formative activity. This is even more clearly confirmed during SEM investigation (Fig. 1E and 1F). The structure of pathological dentin changes during its successive formation and in cases of decrease or cessation of irritation as well as gradual pulp and odontoblast necrosis. In weak and short irritation, changes occurs only in regard to dentinal tubules direction in areas where odontoblasts are damaged (Fig. 4A, detail G, 4B, detail G). Changes in tubules direction in areas where only a few odontoblasts are damaged are even more evident (Fig. 4B, detail G) than in LM samples (3). This is evidence that odontoblast cells react individually thanks to individual defense intracellular potential. It is interesting to notice that, after shallow preparation, at the cavity bottom dentinal tubules are obliterated with amorphous mass similarly to atubular dentin duting pathological dentinogenesis. This implies possible impact of odontoblast processus or dentinal liquor in tubules, as Foreman stated in Other findings also confirm Brannström s theory (6-9) that odontoblast processus are only 0.7 mm from the pulp, implying their role only in the formation of tubules (Fig. 4C and 4D, comparatively). In cases of prolonged irritation such as persisting caries with pulp vitality maintained, pathological dentinogenesis occurs in the same way as after mechanical or chemical irritations (Fig. 2A). The border between regular and pathological dentin is rough with occa-

8 102 Stom Glas S, vol. 53, prekidaju, mestimi no produžavaju unutar patološkog dentina. Broj tubula se prema pulpi smanjuje, a struktura izlu- enog dentina postaje sve iregularnija (Sl. 2A). Uz pulpu se mogu zapaziti zone interglobularnog dentina i jaka proširenja dentinskih tubula (Sl.2B). Ovo ukazuje pre svega na veliku otpornost pulpinih elija da se u dužem vremenskom periodu odupiru dejstvu bakterijskih iritacija, kao i da sturkura patološkog dentina zavisi isklju ivo od stepena ošte enja elija. Ovaj nalaz predstavlja tako e potvrdu slu aja opisanog prilikom analize eksperimentalnih SM izu avanja kada je patološka dentinogeneza u zubu psa, pa ak i kompletna apeksifikacija, obavljena u prisustvu bakterija (3). U slu ajevima nele enog hroni nog karijesa sa kona no avitalnom pulpom, zapaža se tendencija pulpe da se odbrani od bakterijske infekcije slojem skleroziranog dentina. Ova rakcija poti e od strane odontoblasnog niza u celini, pa je i granica sa regularnim dentinom ravna (Sl. 3A, 3B, 3C, 3D detalj K). Me utim, odbrambeni kapacitet je ograni en, odontoblasti pod dejstvom nele neog karijesa, na šta ukazuju i drugi autori (11,12), postupno i pojedina no izumiru i prestaju da lu e tubularni dentin. Granica izme u regularnog i sklerozranog patološkog dentina je stoga neravna. Dentin postaje sve nepravilniji prema kavumu pulpe koja je u fazi izumiranja (Sl. 3A, 3B detalj M). Krajni produkt predstavlja sloj amorfne mineralizvane mase (Sl. 3B- detalj U). Ovakva struktura dentina zapažena je i prilikom apeksifikacije zuba sa nezavršenim rastom korena a u uslovima postepenog izumiranja apikalne pulpe (3). sional discontinuity of dentinal tubules. The number of tubules towards pulp decreases and the structure of dentin becomes more irregular (Fig. 2A). Next to the pulp zones of interglobular dentin and severe dilatation of dentinal tubules can be seen (Fig. 2B). This points out to great resistance of pulp cells to fight bacterial irritation for a long period of time as well as to the fact that structure of pathological dentin depends on the level of cell damage. This result also confirms the results of LM study where pathological dentinogenesis in dog s teeth, even complete apexification, occurred in the absence of bacteria (3). In cases of persisting chronic caries with nonvital pulp, there is a tendency of pulp tissue to defend from bacterial infection with a layer of sclerotized dentin. This reaction comes from the entire odontoblast layer so the demarcation towards regular dentin is smooth and even (Fig. 3A, 3B, 3C, 3D, detail K). However, the defense capacity is limited, odontoblasts gradually die and stop producing dentin in persisting carious infection. This is confirmed by other authors, too (11,12). Border between regular and sclerotized pathological dentin is, therefore rough. Dentin gets more and more irregular towards the pulp chamber in which pulp tissue is becoming necrotic (Fig. 3A, 3B, detail M). The final product is a layer of amorphous mineralized mass (Fig. 3B, detail U). The same dentin structure was noticed during root apexification when apical pulp gradually becomes necrotic (3). Zak.ju ak Skening elektronsko mikroskopska izu avanja prezentirana u ovom radu govore o izuzetno velikoj sposobnosti pulpe zuba da se odupre dejstvu iritacija. Aktivnost o uvanih odontoblasta, kao i onih koji su se kasnije diferencirali da bi zamenili uništene, nije haoti na, ve se odvija po strogo regulisanoj zakonitosti a u okviru interakcije genetski uslovljenih odbrambenih i reparatornih potencijala unutar same elije i dejstva uticaja iz spoljašnje sredine. Na iritativne faktore odontoblasti reaguju individualno ali je mogu e da i pod uticajem jakih iritacija, odontoblasti o uvaju svoj integritet i formativnu aktivnost. Struktura patološkog dentina upravo je srazmerna stepenu ošte enja odontoblasta ili stepenu diferencijacije novih odontoblasta. Postepena diferncijacija novih odontoblasta tako e je razlog za raznovrsnost u strukturi patološkog dentina, koga su formirali u razli itim vremenskim periodima svoje diferencijacije. Rezultati eksperimenta prikazanog u ovom radu, ukazuju da terapija naori ito dubokog karijesa, kao i endodontsko le enje mladih zuba, moraju biti prilago eni zakonitostima koje vladaju u odbrambenom i reparatornom procesu pulpodentinskog kompleksa u celini. Terapija treba da obezbedi uslove za što regularniju formativnu Conclusion SEM study presented in this paper confirms very high capability of pulp tissue to resist irritation. The activity of remaining odontoblasts, as well as those newly differentiated, is not chaotic. It happens highly regularly within interaction of genetic induced defense and reparatory potentials of cells and extrinsic factors. Odontoblasts react individually on irritative factors but it is possible that, even under severe irritation, these cells maintain their integrity and formative activity. The structure of pathological dentin is in relation with the level of odontoblast damage or differentiation of new odontoblasts. Gradual differentiation of new odontoblast cells is also the reason for diversity in structure of pathological dentin which is formed in different time periods of their differentiation. These experimental results imply that therapy of particularly deep caries and endodontic therapy of young teeth must be adapted to regularities in defense and reparatory processes of entire pulp-dentin complex. Therapy should provide conditions for as regular as possible forma-

9 Serbian Dental J, 2006, aktivnost ovih elija, odnosno le enje pulpodentinskog kompleksa treba da omogu i elijama da neometano obave svoj genetski predodre en zadatak u odbrambeno reparatornom procesu. tive activity of these cells. In other words, therapy of pulpdentin complex should enable cells in conducting their genetically predisposed defense-reparatory role without interruption. Literatura / References 1. Vojinovi J, Stevanovi R: Patološka dentinogeneza u posteruptivnoj fazi razvoja zuba. Stom Glas S, 51, 2004: Surdilovi D: Savremena analiza sastava i uloge proteina u dentinu. Magistarska teza, Univerzitet u Nišu, Medicinski fakultet, Stevanovi R, Vojinovi J: Patološka dentinogeneza u posteruptivnoj fazi zuba eksperimentalna svetlosno mikroskopska izu avanja. Stom Glas S, 52, 2005; Foreman J: Structure and Composition of Tubular and Nontubular Deposits in Rout Canal Sistems of Human Permanent Teeth. Int. End J, 21, 27-36, Brannstrom M, Vojinoi O, Nordenvall KJ: Bacteria and Pulpal Reactions Under Silicate Cement Restorations. J Prosther Dent. 41: , Brannstrom M: Dentinal and Pulpal Response. I Aplication of Reduced Pressure to Exposed Dentine. II Aplication of an Air Stream to Exposed Dentine. Short Observation Period. III Aplication of an Air Stream to Exposed Dentine. Long Observation Period. Acta Odont Scoand, 1960, 18:1, 17, Brannstrrom M: The Effect of Dentine Dessication and Aspirated Odontoblasts on the Pulp. J Prosth Dent, 1968, 20: Brannstrom M: A Hydrodinamic Mechanisms i the Transmision of Pain-Producing. Stimuli Tough the Dentine. In: DJ Anderson(ed) Sensory Mechanisms in Dentine. 73 Pergamon. Press Oxford, Brannstrom M: Dentine and Pulp in Restorative Dentistry. Dental Therapeutic, B.A., Nacka, Brannstrom M, Garberoglio R: The Dentinal Tubules and the Odontoblasts Processes. Acta Odnot. Scand, 30: Butler W.T: Dentin Matrix Proteins and Dentinogenesis. Connective Tissue Research, 33, 59-65, Butler W.T, Ritchie H: The Nature and Functional Significanse of Dentin Extracellular Matrix Proteins. Int J Dev Biol., 39: , 1995 Autor odgovoran za korespondenciju Jovan Vojinovi Medicinski fakultet Banja Luka Odsjek za stomatologiju Katedra za de ju i preventivnu stomatologiju Banja Luka BIH Address for correspondence Jovan Vojinovi Faculty of Medicine Banja Luka, Clinic of Dentistry Dept. of pediatric and preventive dentistry Banja Luka BIH