[Construction of cell sheets of stem cells from apical papilla and characterization of their biological properties].

PURPOSE: Cell sheets of stem cells from apical papilla (SCAP) were constructed. Their histological and biological properties were studied to provide experimental basis for its application in dental pulp regeneration in immature permanent teeth.

METHODS: SCAP were isolated and cultured from immature permanent third molars. Flow cytometry was used to evaluate surface marker expression of SCAP. Alizarin red S staining was used to test osteo/odontogenic differentiation capacity of SCAP. SCAP (at P3) were cultured in cell sheets medium for 14 days. Hematoxylin-eosin(H-E) staining was used for histological observation; flow cytometry analysis was used to test cell cycle of SCAP cell sheets; Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and collagen I (Col-I). Western blot was used to detect the protein expression levels of Runx2 and ALP. SPSS 17.00 software package was used for statistical analysis.

RESULTS: H-E staining showed that SCAP cell sheets contained multiple layers of cells (5 to 6 layers) with high cell density, retained tight junctions and secreted rich extracellular matrix. The cell cycle of SCAP cell sheets showed that G2+S phase was lower, while G1 phase was higher than SCAP, which indicated the proliferation rate of SCAP cell sheets was down-regulated (P＜0.05). Moreover, compared with SCAP, osteo/odontogenic differentiation capacity of SCAP cell sheets was significantly increased (P＜0.05).

CONCLUSIONS: SCAP cell sheets secret rich extracellular matrix, and osteo/odontogenic differentiation capacity in SCAP cell sheets is higher than SCAP. SCAP cell sheets may possess more potentials in dental pulp regeneration.