Effects of phosphatidylserine-induced macrophage polarization on the differentiation of human dental pulp cells

Abstract

Macrophages derived from monocytes undergo specific differentiation depending on the local tissue environment. They differentiate into the following distinct functional phenotypes, M1 and M2 macrophages. M1 (classically activated) and M2 (alternatively activated) macrophages are known to play primary roles in inflammation and tissue regeneration, respectively. This study investigated the effects of M1 and M2 macrophages on the differentiation of human dental pulp cells (HDPCs) by using the conditioned media (CM) of the activated human monocyte cell line, THP-1. In addition, the effect of phosphatidylserine (PS), an M2 inducing agent, and PS-treated macrophages on HDPCs differentiation was also assessed. Macrophage polarization was confirmed by flow cytometry analysis targeting surface antigens, and ELISA assay of polarization-related cytokines. To obtain a primary culture of HDPCs, the human dental pulp was extirpated and collected from extracted premolars. To invesigate the feasibility of phosphatidylserine (PS) for polarization of macrophages to M2 phenotype, phosphatidylserine-containing liposomes (PSLs) were used. PS, phosphatidylcholine, and cholesterol were mixed at a molar ratio of 2:1:1, and liposomes were produced via the vacuum evaporation method. To confirm the differentiation of HDPCs, the activity of alkaline phosphatase (ALP), the gene expression of dentin sialophosphoprotein (DSPP) and osteocalcin (OCN), and the degree of matrix mineralization were evaluated. The CM of M2 macrophages (M2CM) enhanced the ALP activity of HDPCs. Furthermore, M2CM promoted the mRNA expression of DSPP gene and matrix mineralization, which indicated that M2 macrophages were able to enhance the differentiation of dental pulp cells. CM of PSLs-treated macrophages enhanced the ALP activity of HDPCs. Furthermore, PSLs were found to promote ALP activity and matrix mineralization of HDPCs. Those results indicate that PSLs can enhance the odontogenic differentiation of dental pulp cells in two different ways

1) via macrophage polarization and 2) promotion of pulp cell differentiation directly. These two effects are exptected to act together on pulp cell differentiation and dentin regeneration.