Uticaj N-acetil-L-cisteina in vitro na proliferaciju i diferencijaciju matičnih ćelija zubne pulpe mlečnih zuba dece

Abstract

Zubna pulpa vodi poreklo od embrionalnog ektomezenhima i potencijalno je vaţan izvor mezenhimalnih matičnih ćelija (MMĆ) za regeneraciju svih tkiva kraniofacijalne regije koja su, takođe, poreklom od ektomezenhima. N-acetil-L-cistein (NAC) je antioksidant koji moţe da ima uticaj na terapijsku primenu MMĆ. Cilj ovog rada je bio da utvrdi da li ćelije izolovane iz zubne pulpe mlečnih zuba dece i ekspandirane in vitro, imaju karakteristike MMĆ i da ispita dejstvo NAC-a na njihovu proliferaciju i diferencijaciju, kao i da se utvrdi da li NAC utiče na aktivnost enzima antioksidativne zaštite, oksidativno oštećenje različitih ćelijskih struktura i metabolizam glukoze. Metodom tkivnog eksplanta, iz šest pulpi mlečnih zuba dece, je dobijena početna populacija adheretnih ćelija. Za procenu broja CFU-F (eng. Colony Forming Unit – Fibroblast), ćelije su zasejavane u maloj gustini. Proliferativni potencijal i vreme udvajanja broja ćelija u kulturi je praćeno brojanjem ćelija posle tripsinizacije subkonfluentnih kultura, svaka 4 dana, tokom 40 dana. Protočna citometrija je korišćena za imunofenotipizaciju ex vivo umnoţenih ćelija, određivanje aktivnosti aldehid- dehidrogenaze (ALDH), brzinu ulaska ćelija u sintetsku (S) fazu ćelijskog ciklusa, određivanje broja ćelijskih deoba i detekciju apoptoze. Aktivnost -galaktozidaze (SA-β-Gal), određivana je korišćenjem citohemije. Diferencijacija ekspandiranih ćelija u smeru osteogeneze, hondrogeneze i adipogeneze izvedena je korišćenjem komercijalnih medijuma. Promene na ćelijama tokom osteogene diferencijacije su praćene preko aktivnosti alkalne fosfataze spektrofotometrijski, deponovanja kalcijuma u ekstracelularni matriks (bojenje alizarin crvenim) i pojave osteokalcina (imunocitohemija). Hondrogena diferencijacija u peletama je praćena određivanjem kolagena tip I (in situ hibridizacija), kolagena tip 2 (imunohistohemija) i određivanjem koncentracije glikozaminoglikana (spektrofotometrija). Adipogena diferencijacija je ispitana vizuelizacijom masnih kapljica (Oil red O bojenje). Aktivnost katalaze i superoksid-dismutaze (SOD) u ćelijskom lizatu je određena spektrofotometrijski. Koncentracija malondialdehida (MDA) je određena reakcijom sa tiobarbiturnom kiselinom (TBA). Karbonilni derivati proteina određivani su reakcijom sa 2,4 -dinitrofenilhidrazinom. Posle ekstrakcije ukupnih lipida i metilovanja masnih kiselina, metil-estri masnih kiselina su razdvajani gasno-tečnom hromatografijom (GLC), a koncentracija zasićenih (SFA), mononezasićenih (MUFA) i i polinezasićenih masnih kiselina (PUFA) je izraţena procentualno u odnosu na ukupne masne kiseline. Izoenzimski oblici laktat-dehidrogenaze (LDH1, LDH2, LDH3, LDH4 i LDH5) su određivani vertikalnom elektroforezom. Sva navedena merenja su izvedena bez i sa različitim koncentracijama NAC-a (0,1 mM, 1 mM i 2 mM). Naši rezultati su pokazali da je iz zubne pulpe mlečnih zuba dece dobijena populacija ćelija koja formira značajan broj CFU-F (4% izolovanih ćelija) i kontinuirano proliferiše tokom 40 dana, bez opadanja vremena potrebnog za udvajanje njihovog broja u kulturi. Ipak, dobijena populacija ćelija je bila heterogena po brzini deoba. Oko 12% ćelija je posle tri dana kultivacije bilo podeljeno manje od četiri puta, dok se ostatak ćelija podelio više od četiri puta. Vijabilitet ćelija je bio u proseku oko 95%, a svega 3% ćelija je bilo pozitivno na β-galaktozidazu. Tokom čitavog vremena kultivacije kapacitet za diferencijaciju u osteoblaste, adipocite i hondrocite se nije menjao. Gotovo sve ćelije su u pasaţi četiri eksprimirale CD44, CD73, kolagen tipa I, CD29, CD90 i osteonektin. Deo ćelija je eksprimirao STRO-1, CD146 i CD106. Marker matičnih ćelija hematopoeze nije detektovan u našem sistemu kultivacije. Opseg u kome se kretao procenat ćelija koje pokazuju povišenu aktivnost ALDH je iznosio od 4% do 24% i bio je u negativnoj korelaciji sa vremenom udvajanja ćelija u kulturi...

Dental pulp originates from the embryonic ectomesenchyme and represents potentially important source of mesenchymal stem cells (MSCs) with the ability to regenerate all tissues of the craniofacial region, originating from the ectomesenchyme, too. N-acetyl-L-cysteine (NAC) is an antioxidant that may have an impact on the therapeutic use of MSCs. The aim of this study was to determine whether the cells isolated from the dental pulp of children deciduous teeth of children and expanded in vitro, have the characteristics of MSC, to examine the effect of NAC on their proliferation and differentiation and to determine whether NAC influences the metabolism of glucose, the activity of antioxidant enzymes and whether it reduces oxidative damage of various cellular macromolecules. The initial cell population was obtained from six pulps of deciduous teeth using ex vivo tissue explants method. The number of colonies (Colony Forming Unit – Fibroblast; CFU-F) was determined by seeding the low density cell culture. Proliferative potential and population doubling time of the cells in culture were followed by counting the cells after tripsinization of subconfluent cultures, every 4 days, respectively, during 40 days of cultivation. Flow cytometry was used for cell immunophenotypisation, aldehyde dehydrogenase (ALDH) activity determination, number of cells in different phases of cell cycle, number of cell divisions and percentage of cells in apoptosis. Activity of β-galactosidase (SA-β-Gal), was determined using cytochemistry. Osteogenesis, chondrogenesis and adipogenesis were induced using complete commercial mediums. Osteogenic differentiation was monitored via alkaline phosphatase activity (spectrophotometry), deposition of calcium in the extracellular matrix (Alizarin red staining) and the appearance of osteocalcin (immunocytochemistry). Chondrogenic differentiation was followed by measuring collagen type I (in situ hybridization), collagen type 2 (immunohistochemistry) and the determination of the concentration of glycosaminoglycans (spectrophotometry). Adipogenic differentiation was examined by visualization of fat droplets (Oil Red O staining). Activity of catalase and superoxide dismutase (SOD) in cell lysates was determined spectrophotometrically. Malondialdehyde (MDA) was determined by reaction with thiobarbituric acid (TBA). Carbonyl derivatives of proteins were determined by reaction with 2,4-dinitrophenylhydrazine. After total lipid extraction and methylation of fatty acids, fatty acid methyl esters were separated by gas-liquid chromatography, and the concentration of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids was expressed as percentage of total fatty acids detected in the sample. Isoenzyme forms of lactate dehydrogenase (LDH1, LDH2, LDH3, LDH4 and LDH5) were determined by vertical electrophoresis. All the above measurements were made with and without different concentrations of NAC (0,1 mM, 1 mM and 2 mM). Our results showed that the dental pulp of deciduous teeth of children had population of cells that formed a significant number of CFU-F (4% of the isolated cells) and continually proliferated for 40 days without decrease in the population doubling time. However, the resulting cell population was heterogeneous in terms of the division velocity. After three days of cultivation, around 12% of the cells were divided less than four times, while the remaining cells divided more than four times. Viability of cells was in average 95%, and only 3% of the cells were positive for β-galactosidase. During the entire time of the cultivation, expanded cells retained the ability to differentiate into osteoblasts, adipocytes and chondrocytes. Almost all cells expressed CD44, CD73, collagen type I, CD29, CD90 and osteonectin. Part of the cells expressed STRO-1, CD146 and CD106. Marker of hematopoietic stem cells have not been detected. Elevated ALDH activity ranged from 4% to 24% and was negatively correlated with the population doubling time...