Cilj ovog istraživanja jest tehnikama proteomske analize potvrditi utjecaj fiksnoga
ortodontskoga aparata na razliku u ekspresiji proteina u proteomskom profilu sline kod
pacijenata u ortodontskoj terapiji u usporedbi s kontrolnom skupinom.
Istraživanje je koncipirano na 18 ispitanika u dobi od 13 godina, muškog spola, bez organskih
oboljenja i zadnje primjene antibiotika unatrag mjesec dana, a kojima je indicirana fiksna
ortodontska terapija. Budući da je proteomski profil sline varijabilan u toj životnoj dobi, prvo
uzorkovanje sline obavljeno je 24 sata prije postavljanja i neposredno pred postavljanje
fiksnoga ortodontskoga aparata. Uzorkovanje sline bilo je nakon 48 sati, 7. dana terapije i 30.
dana terapije te je potom provedena metoda analize uzoraka masenom spektrometrijom.
Analizom dobivenih rezultata kontrolne i terapijske skupine ove studije, najznačajniji nalaz
dobiven je tridesetog dana, a govori u prilog i u skladu je s biologijom koštane pregradnje nakon
primjene ortodontske fiksne terapije u cilju postizanja zubnog pomaka. U svim uzorcima sline
identificirano je 198 proteina te u fazi 30. dana nakon postavljanja ortodontskog aparata
proteom sline sadržavao je dodatnu skupinu proteina koji su uključeni u pregradnju kostiju.
Iz spoznaja do kojih smo došli istraživanjem možemo zaključiti da se pojavnost potvrđenih
molekula: BMP4, BMPER, IGFBP3 i FGF, koja se događa za vrijeme ortodontskog zubnog
pomaka, gotovo identično podudara s pojavnošću molekula prilikom procesa koštane
regeneracije, a koja imitira slijed događanja u embrionalnom razvoju. Od svih koštanih
morfogenetskih proteina navedeno istraživanje identificiralo je molekulu BMP4 u slini koja
zasigurno zauzima važno mjesto u dentalnom razvoju i periodontalnoj regeneraciji. To je prvi
put potvrđena identifikacija molekula BMP-a u uzorku sline. Nadalje, potvrđena je i
identificirana IGFBP3 u slini kao ključna molekula u koštanom metabolizmu. Završno,
identifikacija FGF-a u kontrolnoj i terapijskoj skupini ukazuju nam na njihovu prisutnost u
različitim formama, što bi se moglo povezati i s njihovom protektivnom funkcijom te isto tako
ulogom u regeneraciji i koštanoj pregradnji.
|Abstract (english)|| |
PURPOSE: The aim of this study is to confirm the influence of a fixed orthodontic appliance
on the difference in protein expression in the salivary proteomic profile of patients undergoing
orthodontic therapy compared to the control group, using proteomic analysis techniques.
MATERIALS AND PROCEDURES: The study was conducted on 18 subjects. All subjects
were 13 years old and male, without any organic diseases or a record of antibiotic usage in the
month prior to the study, who are undergoing fixed orthodontic therapy. The number of subjects
in therapy was twelve (N = 12), including six control subjects (N = 6), but without orthodontic
appliances with inclusion and exclusion criteria without risk to patients. Inclusive criteria for
the participants' participation in the research are: age of 13 years, male gender and indication
for fixed orthodontic therapy. The excluded criteria for the participation of respondents in the
research are: existence of some of the systemic diseases that would further alter the affect result
of the research, non-response to control examinations and sudden appearance of the disease
during the research period. There was no risk for the subjects in this study.
Since the salivary proteomic profile in that age group is variable, the first saliva sampling was
performed 24 hours before placement and immediately prior to the placement of the fixed
orthodontic appliance. Saliva was then taken again from all subjects from both groups 48 hours
later, on the seventh day of therapy, and on the thirtieth day of therapy. The saliva collection
procedure was based on three consecutive sputa directly into a Petri dish, after which the saliva
was pipetted into Eppendorf tubes, previously marked with the subjects’ data. Immediately after
the sample collection, the saliva was frozen at -20°C and transported on ice so the samples
could be stored at -80°C.
Finally, the method of sample analysis by mass spectrometry was performed.
RESULTS: The most significant finding in the analysis of all results obtained from control and
therapeutic groups in this study was found on the thirtieth day, and it is in support of and in
accordance with the biology of bone remodeling after the application of fixed orthodontic
therapies used for tooth displacement. From the analysis of isolated proteins obtained in this
study, we can assume the potential role of individual molecules. A total of 198 proteins were
identified in the saliva samples, which is in line with similar saliva studies.
Proteins detected by mass spectrometry were classified on the basis of their functional
characteristics, available in the UniProt database. During the initial and later phase of
orthodontic therapy, we identified proteins involved in neurological processes,
inflammation/stress, cytoskeleton, signal transduction/protein expression, and other processes.
On the 30th day after the placement of the orthodontic appliance, the saliva proteome contained
an additional group of proteins involved in bone remodeling.
CONCLUSION: From the findings of the research, we can conclude that the appearance of
confirmed molecules: BMP4, BMPER, IGFBP3 and FGF, which occurs during orthodontic
tooth movement, almost identically coincides with the appearance of molecules in the process
of bone regeneration that mimics the sequence of events during embryonic development. Of all
the bone morphogenetic proteins, the above study identified the BMP4 molecule in saliva,
which certainly occupies an important place in dental development and periodontal
regeneration. The identification of the BMPER molecule in addition to the BMP4 molecule to
which it clearly binds, confirms the coordinated activity of growth factors and their inhibitors,
which are also confirmed at the proteomic level in the saliva sample as biological material.
Furthermore, IGFBP3 in saliva has been confirmed and identified as a key molecule in bone
metabolism. Finally, FGF identifications in the control and therapeutic groups indicate their
presence in various forms, which could be related to their protective function, as well as their
role in regeneration and bone remodeling.
Considerig that this is the first identification of the BMP molecule (BMP4) in saliva, this result
is extremely important from the aspect of biology of bone morphogenetic proteins, its presence
in biological fluids and the development of new knowledge about how and where BMP4
reached the saliva (locally or systemically through the blood) and further, what is its real role
in orthodontic therapy. To date, proteomic analysis has confirmed the identification of the
BMP6 molecule from the blood and BMP1 (which is not a true BMP molecule, but a
proteinase), wich to is crucial in the activation of BMP molecules and their antagonists.