Determination of alveolar bone loss biomarkers related to peri-implantitis

Uvod. Peri-implantitis predstavlja inflamatorni proces koji se karakteriše gubitkom potporne kosti opterećenog oralnog implantata. Osnovna patološka karakteristika peri-implantitisa je gubitak potporne kosti implantata u funkciji. Ovaj proces je zasnovan na inflamatornoj osteoklastogenezi koja ujedno predstavlja centralni patološki proces peri-implantitisa. Inflamatorna osteoklastogeneza predstavlja proces sazrevanja pre-osteoklasta i pojačavanje aktivnosti zrelih osteoklasta pod uticajem kritičnih koncentracija pro-inflamatornih medijatora. Kliničke karakteristike peri-implantitisa nisu strogo definisane i variraju iz prostog razloga jer dubina peri-implantnog sulkusa značajno varira s'toga dubina džepa predstavlja individualnu determinantu. Istovremeno, proces gubitka marginalne kosti predstavlja fiziološku pojavu koja je najintezivnija u prvoj godini opterećenja, i istraživanja su pokazala da iznosi -0.78mm mezijalno i -0.85mm distalno, a zatim se kontinurano odvija i na godišnjem nivou iznosi oko 0.2mm. Pomenuta vrednost iznosi prosečnu vrednost ali ona takođe individualno varira i uslovljena je tipom implantata, dizajnom abatmenta i mnogim drugim faktorima. Iz tog razloga se relativni nivo pripojnog epitela (rCAL) kao ni radiološki evidentan gubitak kosti ne mogu usvojiti kao apsolutni indikatori patološkog gubitka kosti. U dijagnostici stanja peri-implantnih tkiva koristi se nekoliko tipova metoda i najčešće u kombinaciji radi što potpunijeg postavljanja dijagnoze. Dijagnostičke metode uključuju: određivanje kliničkih parametara, radiološke analize, mikrobiološke analize i kvalitativne i kvantitativne analize peri-implantnte krevikularne tečnosti (PICF). Analiza PICF predstavlja jednu od najatraktivnijih metoda u savremenoj implantologiji, pri čemu je njena najveća vrednost u tome što daje direktne informacije o stanju peri-implantinh tkiva i zasnovano na tome poseduje mogućnost da pokaže rane znake oboljenja peri-implantnih tkiva u fazi gde su tkivne promene reverzibilne. Ovo ograničenje kliničkih metoda rezultira u propuštanju vremena od momenta pojave bolesti koje proporcijonalno umanjuje uspeh terapije, a često i u izboru neadekvatnog terapijskog plana. Zasnovano na tome, metoda merenja specifičnih biomarkera u uzorku PICF nadomešćuje ograničenja konvencionalnih kliničkih dijagnostičkih metoda koje daju informacije u stadijumu razvijene bolesti. Brojne studije se sprovode u cilju identifikacije biomolekula koji pouzdano reflektuje stanje peri-implantnih tkiva, ali kako je patologija lokalnog meatbolizma kompleksna, a metoda evaluacije visoko-osetljiva, standardizacija ove metode je još uvek u toku. Cilj istraživanja bio je da se ispIta potencijal RANK-a, sRANKL-a, OPG-a, Katepsina-K, Sklerostina i VEGF-a kao biomarkera gubitka potporne kosti implantata. Materijal i metode. Studija je obuhvatila tri grupe sistemski zdravih nepušača sa ugrađenim endoesealnim oralnim implantatima, opterećenih tokom najmanje godinu dana (35 sa peri-implantitisom, 30 sa peri-mukozitisom i 30 sa zdravim periimplantnim tkivima). Kriterijum isključenja su bili: upotreba antibiotika u predhodna tri meseca i upotreba antiinflamatorika u predhodna dva meseca od trenutka uzorkovanja, menstrualni ciklus, trudnoća i laktacija, pušenje i tretiranost parodontalnih/peri-implantnih tkiva u poslednjih godinu dana. Klinička merenja su obavljena u 6 tačaka (buko-mezijalna, buko-medijalna, buko-distalna, oro-distalna, oro-medijalna i oro-mezijalna) i uključiće određivanje: krvarenja na probu (BOP) odsustvo-0, prisustvo-1, 15 sekundi nakon sondiranja, indeks akumulacije plaka (PI) odsustvo-0, prisustvo-1 duž marginalne ivice, PD i rNPE graduisanom sondom (North Carolina–Hu-Friedy, Chicago, IL, USA). U slučaju prisustva više peri-implantitisa ili peri-mukozitisa kod jednog pacijenta, zapaljenje sa većim defektom je bilo uključeno u studiju, a u slučaju sličnih karakteristika defekta u 6 tačaka, najdostupnije, odnosno anterijorno mesto je birano kao reprezentativno. Uzorak peri-implantne tečnosti sakupljan je sa mezijalne površine reprezentativnog implantata participanta studije. Uzorci su uzimani 24 časa nakon kliničkih merenja kako bi se izbegla kontaminacija uzorka krvlju, u grupama sa zapaljenjem sa mesta sa najvećom dubinom sondiranja, a u grupi zdravih periimplantnih tkiva sa najdostupnijeg mesta. Uzorci PCF su uzimani sa reprezentativnog mesta metodom filter papira, a dobijeni volumen tečnosti iz tračica je određivan pomoću kalibrisanog aparata Periotron 6000 (Interstate Drug Exchange, Amityville, NY, USA). Komercijalni "enzyme-linked immunosorbent assay" (ELISA) kitovi su korišćeni za evaluaciju koštanih biomarkera u uzorku PICF: Human RANK/TNFRSF11A (DuoSet, R&D Systems Inc., Minneapolis, MN, USA), ampli-sRANKL, OPG, cathepsin-K i sclerostin (Biomedica Gruppe, Vienna, Austria) i VEGF (Human VEGF ELISA Development Kit, Promokine, PromoCell GmbH, Heidelberg, Germany). Rezultati. U svim analiziranim uzorcima PICF-a su dokazane koncentracije RANK-a, sRANKL-a, OPG-a, katepsina-K i VEGF-a iznad detekcionog limita, pri čemu je za sklerostin samo 6% uzoraka bilo pozitivno. Koncentracija RANK-a je bila značajno veća kod peri-implantitisa u odnosu na zdrava periimplantna tkiva (p=0.002), takođe je bila veća i kod peri-mukozitisa u odnosu na zdrave implantate (p=0.021). Vrednosti sRANKL-a bile su značajno veće u grupi peri-implantitisa u odnosu na zdrava peri-implantna tkiva (p=0.010), ali ne i u odnosu na peri-mukozitise, kao ni između peri-mukozitisa i zdravih peri-implantnih tkiva, gde nije postignuta statistička značajnost. Koncentracija OPG-a je bila značajno veća kod peri-implantitisa u odnosu na zdrava peri-implantna tkiva (p=0.031), i to je ujedno bila jedina značajnost za ovaj marker. Vrednosti katepsina-K su bile više na mestima zapaljenja, ali je jedina značajnost uočena između peri-mukozitisa i zdravih peri-implantnih tkiva (p=0.039). Sklerostin je dokazan u izuzetno malom broju uzoraka, ali su razlike bile upadljive pa su vrednosti bile značajno veće u grupi peri-implantitisa u odnosu na druge dve grupe. Koncentracija VEGF-a je bila značajno veća kod peri-implantitisa u odnosu na druge dve grupe, i grupu peri-mukozitisa (p=0.014) i zdravih peri-implantnih tkiva (p=0.000). RANK i sRANKL su pokazali značajno pozitivnu korelaciju sa svim merenim kliničkim parametrima, a OPG je pokazao takođe značajnu pozitivnu korelaciju sa gotovo svim merenim kliničkim parametrima, izuzev sa PI (p=0.121), a identičan slučaj je bio sa sklerostinom. VEGF nije pokazao nijednu značajnu korelaciju sa merenim kliničkim parametrima. Zaključak. RANK, sRANKL, OPG, skelrostin i VEGF su biomarkeri koji su udruženi sa peri-implantitisom. Katepsin-K predstavlja biomarker svosjtven peri-mukozitisu. Evaluirani biomarkeri su drugačije distribuirani u različitim regionima vilica i u PICF implantata različitih dijametara. RANK i OPG su značajno povišeni u frontalnom regionu maksile, što ujedno ukazuje na intezivnije osteolitičke procese u ovom regionu. RANK i katepsin-K su značajno povišeni u grupi implantata sa najvećim dijametrom, što na molekularnom nivou potvrđuje predhodne rezultate kliničkih studija da su dijametar implantata i gubitak implantata u pozitivnoj korelaciji.Introduction. Peri-implantitis is inflammatory process characterized by supporting bone loss of loaded oral implants. The pathognomonic characteristic of peri-implantitis is supporting bone loss of the loaded implant. This process is based on inflammatory osteoclastogenesis which simultaneously represent the central pathologic process of the disorder. Inflammatory osteoclastogenesis implies maturation of pre-osteoclasts and enhancement of the activity of maturated osteoclasts which are induced by achieving of the critical concentrations of proinflammatory mediators. Clinical characteristics of the peri-implantitis are still not strictly defined and they vary because in the physiological conditions the values of clinical parameters varies among individuals, for example peri-implant sulcus depth represents the individual determinant which could be from 0.5mm to 4mm as well. Simultaneously, the marginal bone loss is the physiological characteristic around implants in function, which is the most intensive in the first year of loading represented by the -0.78mm in the mesial sites and -0.85mm at the distal sites, and after that the process is constant and bone loss at the year level is approximately 0.2mm. The mentioned value is the average values that individually vary and it depends of the implant type, abutments and numerous other factors. From that reason the relative clinical attachment level (rCAL), nether radiological proof of bone loss could be accepted as the absolute indicators of the pathological bone loss. In the peri-implant diagnostics the most frequently are used the few different diagnostic procedures in the combination to give the complete diagnostic view. These diagnostic methods include: evaluation of clinical parameters, radiological analyses, microbiological analyses and quantitative and qualitative analyses of PICF. The PICF analysis is one of the most attractive methods in current implantology, where the one of the most precious values is providing of the direct information on peri-implant tissues d based on that providing information on early disease onset in the phase of reversible damage. This limitation of clinical methods results in time loss proportionally decreasing treatment success, and frequently resulting in inappropriate treatment planning. Based on that, evaluation of biomarkers in PICF sample compensates limitations of conventional diagnostic procedures without capability to provide accurate information on early disease. Numerous studies have been conducted to identify the biomolecules accurately reflecting peri-implant tissue condition, but since the pathology of local metabolism is complex, the method for evaluation is still under standardization. Objective. The objective of the study was to investigate potential of RANK, sRANKL, OPG, Cathepsin-K, Sclerostin and VEGF as biomarkers of implant supporting bone loss. Material and methods. Study included three groups of systemically healthy non smokers with osseointegrated endosseal implants loaded for at least one year (35 with peri-implantitis, 30 with peri-mucositis and 30 with healthy peri-implant tissues). Exclusion criteria were the following: antibiotics usage in the preceding three months and anti-inflammatorics in preceding two months from the moment of sampling, menstruation, pregnancy and lactation, smoking and periodontal/peri-implant treatment during last year. The following clinical measurements have been performed in 6 points (bucco-mesial, bucco -medial, bucco -distal, oro-distal, oro-medial and oro-mesial): Bleeding on Probing (BOP) measured 15 seconds after probing and recorded as presence (1) or absence (0), Visible plaque accumulation (PI) measured along the mucosal margin and recorded as presence (1) or absence (0), Probing Pocket Depths (PPD) in mm and Relative Clinical Attachment Level (rCAL) (expressed in mm) using a periodontal probe graded in mm (North Carolina–Hu-Friedy, Chicago, IL, USA). In the case of few similar pathological processes in the same patient, the site representing the greatest defect was sampled, and in the case of defects showing similar clinical characteristics, the most accessible was included. PICF samples were collected from the mesial aspects of one representative implant site in each individual participating in the study. The specimens were retrieved 24 h after the clinical examination to avoid any contamination with blood, from both peri-implant and periodontal sites, selected from those demonstrating the deepest probing depth. The samples were retrieved using the filter paper technique, and obtained volume was evaluated using calibrated Periotron 6000 (Interstate Drug Exchange, Amityville, NY, USA). Commercial enzyme linked immunosorbent kits (ELISA) were used for evaluation of biomarkers in PICF samples: Human RANK/TNFRSF11A (DuoSet, R&D Systems Inc., Minneapolis, MN, USA), ampli-sRANKL, OPG, cathepsin-K i sclerostin (Biomedica Gruppe, Vienna, Austria) i VEGF (Human VEGF ELISA Development Kit, Promokine, PromoCell GmbH, Heidelberg, Germany). Results. In all tested PICF samples were detected RANK, sRANKL, OPG, cathepsin-K and VEGF, indicating the concentrations above detection limit, but only 6% of the samples were positive on sclerostin. RANK concentration was significantly higher in peri-implantitis when compared to healthy periimplant tissues (p=0.002), and it was higher when compared to peri-mucositis as well (p=0.021). sRANKL values were significantly higher in peri-implantitis when compared to healthy peri-implant tissues (p=0.010), but not when compared to peri-mucositis, nether perimucositis an healthy peri-implant tissues. OPG concentration was significantly higher in periimplantitis when compared to healthy peri-implant tissues (p=0.031), and that was single significance obtained for this marker. sRANKL/OPG relative ration did not show significant difference in distribution between investigated groups. Cathepsin-K were in general higher in inflammed sites, but the single significance was reached among peri-mucositis and healthy peri-implant tissues (p=0.039). Sclerostin was detected in small sample size, but the differences were clearly higher in peri-implantitis group when compared to both two groups. VEGF concentration was significantly higher in peri-implantitis when compared to healthy peri-implant tissues (p=0.000) and peri-mucositis as well (p=0.014). RANK and sRANKL showed significantly positive correlation with all measured clinical parameters, and OPG showed significantly positive correlation with all measured clinical parameters as well, with exception of PI (p=0.121), and an identical case was with sclerostin. VEGF showed no significant correlations with clinical parameters. Conclusion. RANK, sRANKL, OPG, sclerostin and VEGF are biomarkers related to peri-implantitis. Cathepsin-K was the marker related to peri-mucositis. Evaluated in this study are differently distributed in different jaws regions and in PICF samples of implants with different diameter. RANK and OPG were significantly elevated in frontal maxillary region, indicating more intensive osteolytic processes in this region. RANK and cathepsin-K were significantly increased in the group o implants with highest diameter, which supports on molecular level the previous results of clinical studies that showed positive correlation between implant diameter and implant loss