Analysis of the influence of acetylsalicylic acid on the differentiation of dental pulp stem cells into mature odontoblasts and the expression of genes responsible for dentin formation in vitro

[M. Vuković]

74 str.

Stomatologija / Dental medicine

Uvod: najnovija istraživanja pokazala su da acetilsalicilna kiselina (ASA), široko korišćen antiinflamatorni lek, poseduje i osteoinduktivno dejstvo. Cilj: da se ustanovi da li ASA može potencijalno da utiče i na odontogenezu, odnosno da stimuliše diferencijaciju matičnih ćelija zubne pulpe (DPSCs) u odontoblaste, i kojim mehanizmima. Materijali i metode: DPSC su izolovane iz tkiva pulpe trećih molara pet pacijenata i uzgajane u osteogenoj podlozi same ili uz dodatak ASA. Ekspresija markera matičnosti testirana je protočnom citometrijom. Citotoksičnost ASA u koncentracijama od 10, 50 i 100 μg/ml testirana je MTT i NR testovima. Osteo/odontogena diferencijacija analizirana je alizarin crvenom bojom i preko aktivnosti alkalne fosfataze (ALP). Kvantitativni PCR je korišćen za analizu ekspresije osteo/odontogenih markera (DSPP, BMP2, BMP4, BSP, OCN i RUNX2), kao i cMYC gena. Rezultati: testovi citotoksičnosti pokazali su da samo najveća doza ASA smanjuje vijabilnost ćelija. Najmanja koncentracija ASA dovela je do značajnog poboljšanja osteo/odontogene diferencijacije DPSC. Došlo je do povećanog formiranja mineralizovanih nodula u ćelijskim kulturama, kao i do porasta aktivnosti ALP i ekspresije svih analiziranih gena (između 2 i 30 puta), u poređenju sa netretiranim ćelijama. ASA je takođe povećala proliferaciju DPSC-a. Nivo ekspresije c-MYC smanjen je u kulturama tretiranim sa ASA, što ukazuje na njegovu ulogu u procesu diferencijacije DPSC ka odontogenoj lozi. Zaključci: pokazano je da niske koncentracije ASA (koje odgovaraju standardnoj upotrebi kod kardiovaskularnih pacijenata) stimulišu osteo/odontogenu diferencijaciju matičnih ćelija zubne pulpe. Klinička važnost: ASA primenjena kao antitrombocitna terapija može potencijalno da pomogne i u reparaciji zubne pulpe.

Introduction: recent research has shown that acetylsalicylic acid (ASA), a widely used anti-inflammatory drug, also has an osteoinductive effect. Objective: to establish whether ASA can potentially influence odontogenesis, i.e. stimulate the differentiation of dental pulp stem cells (DPSCs) into odontoblasts, and by what mechanisms. Materials and methods: DPSCs were isolated from the pulp tissue of the third molars of five patients and grown in osteogenic medium alone or with ASA. Stem cell’s markers expression was tested by flow cytometry. Cytotoxicity of ASA in concentrations of 10, 50 and 100 μg/ml was tested by MTT and NR tests. Osteo/odontogenic differentiation was examined by alizarin red staining and alkaline phosphatase (ALP) activity. Quantitative PCR was used to analyze the expression of osteo/odontogenic markers (DSPP, BMP2, BMP4, BSP, OCN and RUNX2), as well as of cMYC gene. Results: cytotoxicity tests showed that only the highest dose of ASA reduced cell viability. The lowest concentration of ASA led to a significant improvement in the osteo/odontogenic differentiation of DPSCs. There was an increased formation of mineralized nodules in cell cultures, as well as an increase in ALP activity and expression of all analyzed genes (between 2 and 30-fold), compared to untreated cells. ASA also stimulated DPSC proliferation. The expression level of c-MYC was decreased in cultures treated with ASA, indicating its role in the process of DPSC differentiation towards the odontogenic lineage. Conclusions: low concentrations of ASA (corresponding to standard use in cardiovascular patients) were shown to stimulate osteo/odontogenic differentiation of dental pulp stem cells. Clinical relevance: ASA applied as an antiplatelet therapy can potentially help in dental pulp reparation.

matične ćelije zubne pulpe, acetilsalicilna kiselina, osteogena/odontogena diferencijacija, proliferacija, protein kinaza aktivirana adenozin monofosfatom (AMPK)

616.31:577.2(043.3)

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