Cell sheets of human dental pulp stem cells for future application in bone replacement

Abstract

Objectives: To analyze the potential of human dental pulp stem cells (hDPSCs) for maintaining their undifferentiated status and osteogenic differentiation capacity when arranged in cell sheets (CSs) for future application in bone replacement.

Materials and methods: CSs were formed after being induced for 10-15 days by clonogenic medium containing additional vitamin C (20 μg/ml). The cell viability of hDPSC4s in the CSs was followed until 96 h using the Live/Dead® assay. The cells of the CSs were enzymatically dissociated and then compared with the original hDPSC4s. The two cell types were characterized immunophenotypically by flow cytometry using specific mesenchymal stem cell-associated markers (CD105, CD146, CD44, STRO-1, and OCT3/4) and non-associated markers (CD34, CD45, and CD14). Osteogenic differentiation was analyzed with the Alizarin red assay.

Results: Living cells were observed until 96 h in the CSs. Both cell types exhibited osteogenic differentiation and expressed the specific undifferentiated MSC-associated markers. Cells spontaneously detached from the CSs attached and proliferated at the bottom of the culture dishes.

Conclusions: Cells in the hDPSC4s cell sheets survived for at least 96 h. Moreover, the cells in the cell sheets retained their stemness and their osteogenic differentiation potential.

Clinical relevance: Cell sheets of hDPSCs could be employed as natural tri-dimensional structures for treating bone loss. This technique would be useful particularly for critical bone defects or any type of bone defects in patients carrying diseases that impair bone regeneration, such as diabetes mellitus, medication-related osteonecrosis of the jaw (MRONJ), and osteoporosis.

Keywords: Cell sheet; Human dental pulp stem cell; Tissue engineering.