A Pilot Study On Osteogenic Differentiation Abilities Of SHEDs Cell Sheet Induced By Vitamin C In Vitro

Objective:Aim to evaluate the osteogenic differentiation capacity of cell sheets of stem cells from human exfoliated deciduous teeth, stem cell were isolated by enzymes and limited dilution, characterization of stem cells was performed by flow cytometry and multiple differentiation. Stem cell sheets from human exfoliated deciduous teeth were formed by vitamin C induction, the osteogenic differentiation capacities were evaluated by Alizarin Red Staining, alkaline phosphatase staining and relative molecules profiles.Methods:1. Stem cells from human exfoliated deciduous teeth isolation and characterization.Human exfoliated deciduous incisors were collected from6-to10-year-old children from clinic. The pulp tissue was separated from a remnant crown and then digested by enzymatic, single-cell suspensions were cultured in a regular medium as reported. Colony formation rate was observed. The percentage of positive cells was determined, as percentage of cells with expression of the surface antigen CD146, CD90, SSEA-4and OCT-4by flow cytometric analysis. Osteogenesi and adipogenesis were performed and evaluated by Alizarin Red, ALP and oil red O staining.2. Cell sheets formation of stem cells from human exfoliated deciduous teeth induced by vitamin C and osteogenic differentiation observation.The third passage cells were cultured for10-14days in the medium added vitamin C by20,50,80,100μg/ml concentrations. Morphological features by HE staining and osteogenic differentiation by ALP staining and alizarin red staining were observed. The profiles of Runx2and OCN mRNA were detected by RT-PCR.Results:1. Single cell suspensions were capable of forming adherent colonies, characteristic of other stromal stem-cell populations. Some cells formed sphere-like clusters in which highly proliferative cells aggregated together in clusters. The nucleus is round and large, a few contain more than one nucleolus. The colony forming unit was21-24/103cells. Flow cytometric analysis showed SHEDs an average of42.5%positive for CD146,92.51%positive for CD90,41.57%positive for OCT-4and18.3%positive for SSEA-4. Alizarin red-positive nodules formed after14days of osteogenic induction. After4weeks of culture with an adipogenic medium, SHEDs were found to possess the potential to develop into Oil red O-positive lipid-laden fat cells.2. Vitamin C can induce SHEDs to construct high-quality cell sheets at an optimal Vc concentration of50μg/ml. H&E staining revealed that the harvested whole SHEDs sheets was two-or three-layered, and spread uniformly as a two-dimensional tissue structure, which was rich in extracellular. RT-PCR result revealed the mRNA levels of Runx2and OCN were higher in osteogenic medium and vitamin C medium compared to that of control.(P<0.05)Conclusions:1. Acquired stem cells from human exfoliated deciduous teeth by enzyme digestion and limited dilution. SHEDs were identified to be a population of highly clonogenic cells, capable of differentiating into adipocytes, ostetoblasts and so forth. SHEDs express surface markers of mesenchymal stem cells and embryonic stem cells.2. Vitamin C can induce SHEDs to construct high-quality cell sheets at an optimal Vc concentration of50μg/ml.3. SHED sheets are higher capable of the osteogenic differentiation. May be associated with exaltation of the mRNA levels of Runx2and OCN.