Supervisors info:
Αγραφιώτη Αναστασία, Λέκτορας Ενδοδoντίας ΕΚΠΑ, Κοντακιώτης Ευάγγελος, Αναπληρωτής Καθηγητής Ενδοδοντίας ΕΚΠΑ, Χαμπάζ Μαρουάν, Καθηγητής Ενδοδοντίας ΕΚΠΑ
Summary:
Introduction: After completion of root canal disinfection in regenerative
endodontic procedures (REPs), intracanal bleeding creation and blood clot
formation is performed 3-4 mm below the level of cemento-enamel junction. Then,
a few millimetres of coronal barrier are placed upon the formed blood clot and
finally, the tooth is permanently restored. Since plenty of stem cell exist
into the blood clot, coronal barriers are likely to interfere with stem cells.
Therefore, selection of dental material that will be used in regeneration
procedures as coronal barrier is crucial. MTA is the most popular intracanal
barrier as it is refereed in the vast majority of published clinical studies
related to REPs. MTA+Cerkamed has been recently introduced in dental market as
a branch of MTA with improved handling characteristics, decreased setting time
and improved mechanical properties due to the small size of its particles.
TotalFill Bioceramic Root Repair Material (RRM) in putty form has been
developed as a premixed, ready-to-use bioceramic material. TotalFill BC RRM
mainly consists of calcium silicates, calcium phosphate monobasic, zirconium
oxide, calcium hydroxide, tantalum oxide and thickening agents and presents
clinical applications similar to those of MTA. Until now, little information
has been revealed in dental literature on the biocompatibility of MTA+Cerkamed
and TotalFill BC RRM Putty.
Objective: To investigate cytotoxic effects of TotalFill BC RRM and
MTA+Cerkamed in comparison to ProRoot MTA on stem cells from human exfoliated
deciduous teeth (SHED) and to evaluate the cell morphology on surfaces of these
materials.
Materials & Methods: SHED were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM), supplemented with streptomycin, penicillin, and L-glutamine and
complemented with 10% heat-inactivated fetal bovine serum at 37oC in a 5% CO2
humidified incubator. Cells used were between 3rd and 5th passage. TotalFill BC
RRM-Putty, ΜΤΑ+Cerkamed and ProRoot MTA were prepared and were placed on 13-mm
cover slips where they were allowed to set for 2 hours. After that, the dental
materials were transferred in the bottom of the wells of a 24-well culture
plate with 300 μl DMEM. Wells without any endodontic material served as
control. Cytotoxicity assay was performed by using transwells. 300 μl of DMEM
was added in each transwell and 104 SHED were seeded on the porous,
polycarbonate membrane of the transwells. Due to the size of the porous, the
cells were unable to move through the membrane. They were cultured for 72
hours, trypsinised, collected, stained with trypan blue and counted using
Neubauer hematocytometry. Kruskal Wallis test was performed along with post-hoc
analysis with the use of Mann-Whitney test for investigating statistically
significant differences between each potential pair of materials. All reported
probability values (p-values) were compared to a significant level of 5%. The
analyses of coded data were carried out using IBM SPSS software version 21.0.
To evaluate the cell morphology, twelve intact, fully developed, single-rooted
human mandibular premolars that were extracted for orthodontic reasons were
recruited. A root segment of 3mm in thickness was collected from the middle
part of each root. Root canals were enlarged to 1.1 mm in diameter by using
Gates Glidden drills #1 to 4. The dental materials were prepared and packed
into the lumen of each root segment (4 root segments were used for each
material). Each specimen was placed in the bottom of the well of a 24-well
culture plate. 300 μl DMEM and 2.5x104 cells were added in each well. The cells
were cultured for 72 hours at 37oC in a 5% CO2 humidified incubator. After
that, the cells cultured on the surface of the samples were fixed and the
samples were stained using Phalloidin-Rhodamin solution to reveal the
cytoskeleton and more specifically the actin filaments. 4’,
6-diamidino-2-phenylindole (DAPI) was used for the detection of the nucleus.
Samples were observed under the confocal microscope Leica TCS SP5 and analyzed
using Leica software, LAS AF.
Results: Expressed as percentages of control group, the mean values of viable
cells in 3 groups (TotalFill BC RRM, MTA+Cerkamed and ProRoot MTA) were 240%,
100% and 140% respectively. The mean value of viable cells in TotalFill BC RRM
group was significant higher than any other group (p<.05). Cells on surfaces of
TotalFill BC RRM and ProRoot MTA demonstrated a spindle-shaped morphology with
thin, multiple processes, whereas on surfaces of MTA+Cerkamed, they were more
retracted, displaying a rounded-shaped morphology.
Conclusion: TotalFill BC RRM demonstrates considerably high level of cell
viability. The level of cell viability for MTA+Cerkamed was satisfactory and
acceptable. The surface of MTA+Cerkamed was not found to be as welcoming as
that of TotalFill BC RRM and ProRoot MTA. The in vitro biocompatibilty of
TotalFill BC RRM is noteworthy and encourages the application of this material
in clinical practice.
Keywords:
Biocompatibility, ProRoot ΜΤΑ, MTA+Cerkamed, Regenerative Endodontics, Stem cells
