The patterns of proliferation in a human tooth germ during the fetal developmental period

Cilj istraživanja: Cilj istraživanja je bio prikazati obrasce proliferacijske aktivnosti stanica u zametcima ljudskog zuba tijekom razvojnih stadija pupoljka, kape i zvona. Materijali i metode: U svrhu istraživanja korišteni su uzorci tkiva uzeti iz deset ljudskih fetusa starih 7, 8/9, 10 i 14 tjedana trudnoće dobivenih nakon spontanih pobačaja i ektopičnih trudnoća te preuzetih s Odjela patologije KBC-a Split. Imunohistokemijska analiza provedena je na fetalnom tkivu u području dijelova čeljusti koji su sadržavali zubne zametke. Rezovi su bojani primarnim monoklonskim protutijelima za Ki67 i p19. Preparati su fotografirani digitalnom kamerom postavljenom na fluorescencijski mikroskop, a dobivene slike su obrađene u AdobePhotoshop® CS6. Superponiranjem slika (merge) te obrađivanjem istih u ImageJ softveru procijenjivala se izražajna domena ispitivanih čimbenika. Rezultati: Proliferacijski aktivne stanice pozitivne na Ki67 uočene su u svim ispitivanim stadijima zubnog zametka. Zastupljene su podjednako u epitelnim i mezenhimnim dijelovima zubnog zametka, a početkom stadija zvona (histodiferencijacija) su bile najbrojnije u njegovim epitelnim dijelovima, odnosno unutarnjem caklinskom epitelu, stratum intermediumu i cervikalnim petljama. Izražajna domena p19 se uvelike preklapala s tkivima zubnog zametka u kojima su se nalazile stanice pozitivne na Ki67 u svim ispitivanim stadijima razvoja, ali je u stadiju zvona p19 bio izražen i u mezenhimnim dijelovima zubnog zametka (dentalna papila). Zaključak: S obzirom na postojeće znanje o osnovnoj funkciji p19 i rezultatima dobivenim u ovom istraživanju, može se zaključiti da p19 istovremeno sudjeluje u procesima proliferacije i diferencijacije u zametku zuba tijekom njegovog ranog razvoja te da je aktivnost p19 važno molekularno obilježje interakcija između epitelnih i mezenhminih dijelova zametka ljudskog zuba.Objectives: The goal of research was to show patterns of cell proliferative activity in developing human tooth germ during bud, cap and bell stages. Material and Methods: For the purpose of the study, tissue samples were taken from ten human fetuses aged 7, 8/9, 10 and 14 weeks of gestation after spontaneous abortions and ectopic pregnancies from University Hospital Split Department of Pathology. Immunohistochemistry analysis was performed on fetal tissues from head area or parts of the jaw containing tooth germs. Tissue sections were stained with primary monoclonal antibodies against Ki67 and p19. Stained histological tissue sections were photographed by digital camera mounted on a fluorescence microscope, and the microphotographs were processed in AdobePhotoshop® CS6. Merged image doublets were then assembled and processed by using ImageJ software, to evaluate the expression domains of the examined factors. Results: Ki67 positive proliferating cells were observed in tissues of human tooth germ throughout the investigated period. Ki67 proliferating cells were distributed equally in the epithelial and mesenchymal parts of the tooth germ, whereas during the early bell stage (histodifferentiation) these cells were more localized in its epithelial parts, such as the inner enamel epithelium, stratum intermedium and cervical loops. Expression domains of p19 greatly overlapped with areas populated by Ki67 positive proliferating cells throughout the investigated period, but at the bell stage p19 was also expressed in the mesenchymal parts of the dental germ (the dental papilla). Conclusion: Based on the present knowledge about p19 cellular functions and results from this study, it might be suggested that p19 can simultaneously participate in proliferation and in differentiation processes during the early tooth development and that p19 activity is an important molecular feature of epithelial-mesenchymal interactions regulating basic cellular processes in developing human tooth germ