Neural Differentiation of Wisdom Tooth Follicle Stem Cells on a Nano-Hydrogel Scaffold Containing Salvia Chloroleucat to Treat Nerve injury in the Cancer of Nervous System

Abstract

The human third molar's follicle is one of the sources of stem cells with high differentiation capacities which can be used in nervous system cancer treatment particularly in nerve damge. The purpose of this research was to identify the effects of the aqueous extract of Salvia chloroleuca on the differentiation of the human dental follicle-derived mesenchymal stem cells to neural cells for treti. In this experimental study, the method of culture of digested tissue fragments was used to isolate stem cells from three samples of the extracted wisdom teeth follicles. The nano-hyaluronic acid scaffold has been synthesized by the sol-gel method as a porous composite and the S. chloroleuca extract has been loaded into it. The scaffold was analyzed in terms of mechanical properties, drug release and toxicity. Afterwards, the cells were seeded onto the scaffold using the immersion method. After 21 days, cell differentiation was investigated by morphological confirmation methods and confirming the expression of β-tubulin and MAP2 genes at mRNA and protein levels. Morphological assessment revealed neural differentiation in the cells of the groups of nano-hyaluronic acid scaffold with S. chloroleuca extract and nano-hyaluronic acid scaffold with S. chloroleuca extract + 10% retinoic acid. Furthermore, the expression of MAP2 and β-tubulin in these groups was confirmed by RT-PCR, real time PCR and western blot assays. The results of this research showed that the follicle of the third molar contains stem cells with a high capacity for differentiation. Moreover, the extract of S. chloroleuca, could lead to induction of neural differentiation in stem cells.

Keywords: S. chloroleuca; Stem Cell; follicle; neural differentiation; wisdom tooth.

Figure 1

Differentiation Potential to Adipocyte with Oil Red Staining (A) and osteoblasts with Alizarin Red staining (B)

Figure 2

Flow Cytometry Histogram of the Expression of Mesenchymal and Hematopoietic Markers with the Control Isotype Sample

Figure 3

Evaluation of the Synthesized Scaffold Containing S. chloroleuca Extract (A: SEM microscope images of nano-hyaluronic acid scaffold, B: MTT diagram in 72 hours for nano-hyaluronic acid scaffold, C: drug loading in nano-hyaluronic acid scaffold, D: drug release of nano-hyaluronic acid scaffold, E: physical properties of nano-hyaluronic acid scaffold and F: results of nano-hyaluronic acid scaffold mechanical tests)

Figure 4

Morphological Evaluation of Cultured Cells in Five Studied Groups, Including Stem Cells in Standard Neural Culture Medium (A), negative control cells cultured in DMEM+FBS 10% medium (B), stem cells seeded on nano-hyaluronic acid scaffold without S. chloroleuca extract (C), stem cells seeded on nano-hyaluronic acid scaffold with S. chloroleuca extract (D), stem cells seeded on nano-hyaluronic acid scaffold with S. chloroleuca extract +10% retinoic acid (E)

Figure 5

Evaluation the Expression of MAP2 and β-Tubulin Genes in the Human Dental Follicle-Derived Mesenchymal Stem Cells after 14 Days of Culture in Different Groups by qRT-PCR Method (A), Real time PCR (B: MAP2 and C: β- Tubulin) and western blot (D: MAP2 and E: β-Tubulin). In all figures, column 1: the nano-hyaluronic acid scaffold without S. chloroleuca extract, column 2: the nano-hyaluronic acid scaffold containing S. chloroleuca extract, column3: the nano-hyaluronic acid scaffold containing S. chloroleucaextract + 10% retinoic acid, column 4: normal culture medium without the presence of differentiation factors, column 5: standard neural culture medium with differentiation factors, column 6 (NT2): positive control group containing NT2 cells. M: 100 bp DNA ladder