الفهرس 
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Abstract
In thepast two decades, the endodontic dental materials have been widely improved. Therefore, it was important to continuously investigate and compare their biological behavior.Several studies applied the principles of tissue engineering using mesenchymal stem cells in evaluatingthe endodontic materials bioactivity and biocompatibility.
The aim of the present study was to evaluate and comparethe effect of different bioactive endodontic materials namely;Nano hydroxyapatite (NHA), Mineral trioxide aggregate (MTA) and Calcium enriched mixture (CEM)on the human dental pulp stem cellsin vitro regarding;odontogenic differentiation, proliferation and viability to assess theirbioactivity and biocompatibility.
• Materials and methods:
Populations of dental pulp cellswere isolated usingenzyme-digestion method from human vital third molar teeth and expanded in-vitro with culture growth medium (GM)via cell culture techniques.The cultured dental pulp cells werecharacterized bymonitoring their growth, self-renewal capability and colony forming efficiency during the culture period.Moreover, the relative expression of stem cell genes in the cultured cells was evaluatedusing quantitiveRT-PCR test.
To evaluate the effect of the tested biomaterials on the cultured dental pulp stem cells, all biomaterial were mixed according to manufacturer instructions and shaped into cylinders.Then the biomaterial cylinders were incubated inCO2incubator at 37°C and humidified atmosphere100%for 24 hours to ensure complete setting,and then sterilized using ultra violet light.Populations from the thirdand fourthpassages of the cultured cells weretrypsinized and seeded into 6-wells culture plates at a density of 15x103 cells per 3mlfreshly prepared growth culture medium (GM) in each well.
To evaluate the effect of the tested biomaterialsdirect contact on the odontogenic differentiation of dental pulp stem cells,these cells were incubateduntil reached 70% confluencythen they were groupedinto four groupsas follows:
• Group(I):the confluent monolayers of DPSCs were incubated withodontogenic differentiation medium (DM)(odontogenically inducedDPSCs).
• Group(II): the confluent monolayers of DPSCs were incubated with growth culture medium (GM)(un-induced DPSCs)
Then both groups I &II were further subdivided into three subgroups (A, B and C) according to the biomaterial (NHA, MTA and CEM respectively)used to supplement the DPSCs.
• group (III) (positive control):wells with no biomaterials supplemented to odontogenically induced DPSCs with DM were used as positive control group.
• group (IV)(negative control): wells with no biomaterials supplemented to un-induced DPSCs with GM were used as negative control group.
To inspect the effect of biomaterials on the odontogenic differentiationof DPSCs, cells were monitored by inverted phase contrast light microscope for 14 days. After that an alizarin red stainingwas carried out and the relative expressions of odontoblastic differentiation genes were measured by quantitivereal-time reverse-transcription polymerase chain reactions (qRT-PCR).
In addition, the effect of the tested biomaterials direct contact on the proliferation and viability of cultured dental pulp stem cells was evaluated. Thetrypsinized cells from thirdand fourthpassages werefirst incubated for 24 hours in culture platesto allowfor cell rest andattachment then, the plates were grouped as follows;
• group (I):un-induced DPSCs with GM were subdivided into three subgroups (A, B and C) according to the biomaterial (NHA, MTA and CEM respectively)used for supplementation.
• group (II):induced DPSCs were cultured withodontogenicdifferention medium (DM).
• group (III):un-induced DPSCs cultured with GM without biomaterials supplementation (control group).
DPSCs in all groups and subgroups were then cultured and monitored by inverted phase contrast light microscope at the same time intervals; for 1, 3, 5, 7, 9, 11 and 14 days. At these intervals cell counts and viabilities were measured using trypan blue exclusion method.
All experiments were done in triplicates to assure reproducibility and the data obtained recorded for statistical analysis using SPSSversion (20) software program.
• Results:
The results showed that the isolated human dental pulp cells were including stem cells populations that had a positive self-renewal capability and colony forming efficiency. Furthermore the isolated cells expressed stem cell genes at the third and tenth generations.
Regarding the evaluation of odontogenic differentiation, all tested biomaterials were foundto promote the odontogenic differentiation of un-induced DPSCs(DPSCs in growth medium)in a similar manner to that of odontoinduction media. Additionally these biomaterials showed no statistical significant difference in odontogenic genes expression with each other and with odontogenic induction media. However these biomaterials when cultured with induced DPSCs (DPSCs in odontogenic induction media at the same time)showed more odontogenic differentiation potential (in terms of matrix mineralization and odontogenic genes expression) than those biomaterials cultured with uninducedDPSCs or than DPSCs cultured with odontogenic induction medium only.
According to the proliferation (cell count) and viability results of culturing DPSCs in direct contact with the tested biomaterials,there was an initial significant decrease in all biomaterials supplemented DPSCscounts and viabilities at day one in comparison to the odontogenic differentiation medium (DM) and control group (GM). At the same time, no significant differences between the tested biomaterials were noticed at that day.Then this decrease in cell counts and viabilities discontinued andinstead a significant rise was noted starting from day three. This rise in cell counts and viabilitiesreached its peak at day five for the NHA,at day sevenfor CEMand DMsupplemented cells.Whereas, the MTA supplemented cells reached its peak in the cell counts and viabilities at day eleven and seven respectively.
The MTA supplemented cells showed non-significantly slight more counts and viabilities than CEM at all day intervals, except at day nine wherethe MTA supplemented cells showed significantly greater cell viability.Whereas, there was a general decreasein the mean count and viabilities in the NHA supplemented cells in comparison to other tested biomaterials that was significant after day five.
DPSCs that were cultured in direct contact with MTA, CEM and DM proliferated in a similar manner. Moreover, the cells proliferated at the MTA and CEM cylinders were with normal appearance and limited signs of necrosis/apoptosis or cell free zones in the culture especially at and before day nine. On the other hand, DPSCs cultured with NHAshowed noticeable signs of necrosis/ apoptosis and cell free zones surrounding the NHA cylinders in almost all culture plates especially after day five.