Ozon u restaurativnoj stomatološkoj terapiji: antibakterijski efekat i uticaj na VEGF u zubnoj pulpi/

Abstract

Uvod. Ozon ima široku primjenu u medicini, a posljednjih godina se primjenjuje u stomatologiji zbog izraženog antimikrobnog djelovanja bez pojave antimikrobne rezistencije. Manji broj kliničkih studija ukazuje na mogućnost primjene ozona u terapiji karijesnih lezija i za dezinfekciju kaviteta. Za odontoantiseptike je od značaja njihov farmakodinamski efekat kao i njihov bezbjednosni profil. Najveći broj do sada objavljenih radova o antibakterijskoj efikasnosti ozona na kariogene bakterije odnosi se na rezultate in vitro studija, a mali broj radova na ispitivanje biokompatibilnosti ozona na zubnu pulpu in vivo. Cilj. Cilj ove teze je bio da se ispita antibakterijski efekat primjene ozona na ukupan broj bakterija, Lactobacillus spp. i pojedine vrste Lactobacillus spp. poslije nepotpunog uklanjanja karijesa, kao i da se odredi koncentracija vaskularnog endotelnog faktora rasta (VEGF) i superoksid dismutaze (SOD) u zubnoj pulpi zdravih zuba poslije primjene ozona. Materijal i metode. Studija I je uključila ukupno 48 zdravih osoba oba pola, starosti od 19-48 godina, kod kojih je dijagnostikovana najmanje jedna duboka karijesna lezija (>1/2 debljine dentina) na okluzalnoj ili okluzalnoj i aproksimalnoj površini bočnog zuba. Ispitanici su u zavisnosti od primjenjenog antiseptika za dezinfekciju kaviteta podijeljeni u dvije grupe: eksperimentalna, u kojoj je korišćen ozon u obliku gasa, i komparativna grupa, gdje je primjenjen 2% hlorheksidin. Uzorci dentina su uzeti iz karijesnih kaviteta poslije nepotpunog uklanjanja karijesa i poslije primjene antiseptika i analizirani su kultivisanjem, lančanom reakcijom polimeraze (PCR) i „real-time“ PCR (qPCR). Studijom II su obuhvaćene 38 zdrave osobe oba pola, starosti 18-40, kod kojih je indikovana ekstrakcija intaktnih i klinički zdravih donjih molara. Poslije preparacije kaviteta na okluzalnoj površini zuba u eksperimentalnoj grupi je primjenjen ozon u obliku gasa, dok u kontrolnoj grupi kaviteti nisu tretirani (fiziološki rastvor). U zubnim pulpama određen je nivo VEGF imunoesejskom tehnikom (ELISA) i nivo SOD spektrofotometrijskom metodom. Rezultati. Studija I. Jednokratna primjena ozona i hlorheksidina u preparisani kavitet je dovela do značajnog smanjenja ukupnog broja aerobnih i anaerobnih bakterija, Streptococcus spp., Lactobacillus spp., ali ne i pojednih vrsta Lactobacillus spp., nezavisno od metode identifikacije bakterija. Komparativna analiza antibakterijskog efekta ozona i hlorheksidina ispitivana metodom kultivisanja je pokazala da je ozon izazvao značajno smanjenje broja Lactobacillus spp. (p=0,041), a hlorheksidin značajno smanjenje broja Streptococcus spp. (p=0,041). Primjenom PCR i qPCR metode nije zabilježena razlika u efikasnosti ozona i hlorheksidina na kariogene bakterije. Studija II. Koncentracija VEGF u grupi tretiranoj ozonom je bila značajno veća (p<0,0001), a nivo SOD značajno manji (p=0,002) u odnosu na vrijednosti dobijene za pulpe kontrolnih zuba. Zaključci. Komparativna analiza rezultata o antibakterijskoj efikasnosti jednokratno primjenjenog ozona i 2% hlorheksidina u ispreparisani kavitet zuba pokazuje da su oba antiseptika podjednako efikasna. Poslije jednokratne primjene ozona kao antiseptika u kavitet zuba povećava se nivo VEGF, koji ne predstavlja patološko povećanje, i aktivira se antioksidativni sistem u tkivu pulpe, što ukazuje na njegovu biokompatibilnost sa zubnom pulpom.

Introduction. Ozone is widely used in medicine, and recently it is used in dentistry due to its strong antimicrobial activity without antimicrobial resistance. A small number of clinical studies evaluated the effect of ozone in therapy of dental caries and for cavity disinfection. For dental antiseptics use, both their pharmacodynamic effects and safety profile are of the importance. Most of published works considering the antibacterial effect of ozone on cariogenic bacteria has been conducted in vitro, and a small number of studies investigated biocompatibility of ozone on dental pulp in vivo. Aim. The aim of this thesis was to investigate the effect of ozone on total bacteria, Lactobacillus spp., and different Lactobacillus spp. strains after incomplete caries removal, as well as to determine the concentration of vascular endothelial growth factor (VEGF) and superoxide dismutase (SOD) in the dental pulp of healthy teeth after ozone application. Material and methods. Study I included 48 healthy subjects of both gender, aged 19-48 years, with at least one tooth with diagnosed deep caries (> 1/2 of the thickness of dentin) on occlusal or occlusal and interproximal tooth surface. Subjects were divided into two groups according to the cavity disinfectant used: the experimental group, where ozone gas was used, and the comparative group, where 2% chlorhexidine was used. Dentine samples were taken from the cavity after partial caries removal and after application of antiseptic, and analyzed using cultivation, polymerase chain reaction (PCR) and "real-time" PCR (qPCR) methods. Study II included 38 healthy subjects of both gender, aged 18-40 years, in which the extraction of sound mandibular molars were indicated. After cavity preparation on the occlusal surface of the tooth ozone gas was applied in the experimental group, while saline was applied in the control group. The levels of VEGF and SOD in dental pulp were determined by immunoassay technique (ELISA) and by spectrophotometric method, respectively. Results. Study I. Single application of ozone and chlorhexidine into the cavity resulted in a significant decrease of the total number of aerobic and anaerobic bacteria, Streptococcus spp., and Lactobacillus spp., but not of the Lactobacillus spp. strains, irrespective of the method for bacteria enumeration. Comparative analysis of antibacterial effect of ozone and chlorhexidine using cultivation revealed that the number of Lactobacillus spp. was significantly lower in the ozone group while the number of Streptococcus spp. was significantly lower after chlorhexidine treatment (p=0.041). By using PCR and qPCR methods, no difference in the effectiveness of ozone and chlorhexidine on cariogenic bacteria was noted. Study II. In the group treated with ozone the concentration of VEGF was significantly higher (p<0.0001), and the level of SOD was significantly lower (p=0.002) compared to the values obtained for the control pulps. Conclusions. Comparative analysis of the antibacterial efficiency of once applied ozone and 2% chlorhexidine in tooth cavities indicates that both antiseptics are equally effective. After single application of ozone in tooth cavity the level of VEGF in dental pulp increases, which does not present pathological increase, and the antioxidant system activates, which indicates its biocompatibility with dental pulp.