الفهرس 
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Abstract
Introduction:
Regenerative endodontic procedure is a novel modality which involves physiological replacement of the damaged structures. This procedure considered the treatment of choice for necrotic immature permanent teeth because it promotes the completion of root development, thickening of their weak dentinal wall and normal physiologic responses. However, this procedure is based on the combination and interplay of 3 key elements for tissue regeneration, namely stem cells, bioactive molecules, and scaffolds. (1)
Stem cells of the apical papilla are derived from an embryonic neural crest like soft tissue, located at the apex of the incompletely developed root(71). One of the most important aims in regenerative endodontic treatment is to create a healing environment for regeneration so that sealing material can be placed on top of the blood clot. Thus, biocompatible sealing materials are essential. The sealing material should prevent bacterial or toxin leakage, show biocompatibility, and enhance cell proliferation and differentiation. (48)
Aim of the study:
The aim of the study was to evaluate the effect of Hydroxyapatite coated by Chitosan Nano-particles and Bioactive Glass Nano-particles on osteogenic differentiation and proliferation of stem cells of apical papilla.
Materials and methods:
First of all, preparation and characterization of the tested nano materials was done. For in vitro testing, three fully impacted immature third molars were used in the study. SCAP were isolated using enzymatic digestion method.
Then, characterization was done using flow cytometric analysis. After characterization, SCAP were cultured in complete culture media. All tested nanomaterials were mixed according to manufacturer instructions. The samples were classified to five equal groups:
• Negative control group: SCAP with DMEM.
• Positive control group: SCAP with inductive media (OM).
• group I: Nanohydroxyapatite [NHAP 10ug/mL] with SCAP.
• group II: Nanohydroxyapatite coated by chitosan [NHAP/Ch 10ug/mL] with SCAP.
• group III: Bioactive Glass Nanoparticles [NBG 500ug/mL] with SCAP.
For osteoblastic differentiation, SCAP were cultured OM seeded at 4.5 × 105 cells/well (6-well plates). Plates were incubated at 37°C and 5% CO2 for 72 hours, and then different tests for differentiation and proliferation potentials was applied. The activity of ALP was measured using enzymatic dephosphorylation by ALP assay kit. For testing the expression of RANKL for SCAP, the cells were examined using specific polyclonal antibody by Fluorescence microscope.
Regarding evaluation of the proliferation, the SCAP were stained by trypan blue and counted by hemocytometer to estimate the number of dead cells. The MTT assay was performed using the Vybrant® MTT Cell Proliferation Assay Kit. Cell viability was determined by measuring the optical density at 570 nm on a spectrophotometer.
Results:
The observed results of the characterized SCAP, revealed that the cells showed double bright surface expression of CD44/CD73 and didn’t express CD45, which confirm that the isolated stem cells are isolated from non-hematopoietic source.
Regarding ALP assay results, NHAP/Chitosan showed the highest concentration of AlP followed by NBG followed by NHAP and DMEM-NC showed the lowest concentration. There was a statistically significant difference between all tested groups. While, RANKL expression results, NHAP/Chitosan showed the highest H score followed by NBG followed by NHAP and DMEM-NC showed the lowest H score. There was a statistically significant difference between all tested groups.
Regarding Viable cell count, NHAP/Chitosan showed the highest cell count while DMEM-NC showed the lowest cell count. While the [OM-PC] and [DMEM-NC] groups showed statistically significant difference with the other three tested groups; There was no statistically significant difference between each other. While, MTT assay results, the [OM-PC] and [DMEM-NC] groups showed statistically significant difference with the other three tested groups, there was no statistically significant difference between them.