Changes in characteristics of periodontal ligament stem cells in spheroid culture.

OBJECTIVES: The periodontal ligament (PDL) has important roles in maintaining homeostasis, wound healing, and regeneration of periodontal tissues by supplying stem/progenitor cells. Periodontal ligament stem cells (PDLSCs) have mesenchymal stem cell (MSC)-like characteristics and can be isolated from periodontal tissues. The aim of this study was to examine the effect of three-dimensional spheroid culture on the characteristics of PDLSCs.

MATERIAL AND METHODS: Periodontal ligament stem cells were isolated and cultured from healthy teeth, and PDLSC spheroids were formed by pellet culture in polypropylene tubes. The proliferation of PDLSCs in spheroids and conventional two-dimensional (2D) cultures were examined by immunostaining for Ki67. Cell death and cell size were analyzed using flow cytometry. Gene expression changes were investigated by quantitative real time PCR.

RESULTS: Periodontal ligament stem cells spontaneously formed spheroid masses in pellet culture. The size of PDLSC spheroids was inversely proportional to the culture period. Fewer Ki67-positive cells were detected in PDLSC spheroids compared to those in 2D culture. Flow cytometry revealed an increase in dead cells and a decrease in cell size in PDLSC spheroids. The expression levels of genes related to anti-inflammation (TSG6, COX2, MnSOD) and angiogenesis (VEGF, bFGF, HGF) were drastically increased by spheroid culture compared to 2D culture. TSG6 gene expression was inhibited in PDLSC spheroids in the presence of the apoptosis signal inhibitor, Z-VAD-FMK. Additionally, PDLSC spheroid transplantation into rat periodontal defects did not induce the regeneration of periodontal tissues.

CONCLUSIONS: We found that spheroid culture of PDLSCs affected several characteristics of PDLSCs, including the expression of genes related to anti-inflammation and angiogenesis; apoptosis signaling may be involved in these changes. Our results revealed the characteristics of PDLSCs in spheroid culture and have provided new information to the field of stem cell research.