Hee Chul Park1, Hongxuan Quan1, Tingting Zhu1, Yongjoon Kim1, Bongju Kim2, Hyeong-Cheol Yang3. 1. Department of Dental Biomaterials Science and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea. 2. Dental Life Science Research Institute, Seoul National University Dental Hospital, Seoul, Korea. 3. Department of Dental Biomaterials Science and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea. Electronic address: yanghc@snu.ac.kr.
Abstract
INTRODUCTION: M1 (classically activated) and M2 (alternatively activated) macrophages are known to play primary roles in inflammation and tissue regeneration. To investigate the role of macrophages in dentin regeneration, this study examined the effects of M1 and M2 macrophages on the odontogenic/osteogenic differentiation of human dental pulp cells (HDPCs) using the conditioned media (CM) of the activated human monocyte cell line THP-1. METHODS: M1 and M2 macrophages were induced by lipopolysaccharide/interferon-γ and interleukin-4, respectively, and the phenotypes were confirmed by flow cytometry. Macrophage CM was prepared at 2-day intervals for a period of 6 days, which included the first 2 days of activation. The CM obtained on days 4 (M1CM-4 day and M2CM-4 day) and 6 (M1CM-6 day and M2CM-6 day) were tested for their ability to promote the alkaline phosphatase (ALP) activity of HDPCs. M2CM-4 day was also examined for its effects on the messenger RNA expression of dentin sialophosphoprotein and osteocalcin genes and the matrix mineralization of HDPCs. Tumor necrosis factor alpha and transforming growth factor beta 1 (TGF-β1) in M1CM and M2CM, respectively, were quantified by an enzyme-linked immunosorbent assay. To verify the role of TGF-β1, M2CM-4 day was pretreated by a TGF-β blocking antibody and was examined for its effect on the ALP activity of HDPCs. RESULTS: M2CM-4 day and M2CM-6 day enhanced the ALP activity of HDPCs (P < .05). Furthermore, M2CM-4 day promoted the messenger RNA expression of the dentin sialophosphoprotein gene and matrix mineralization (P < .05), whereas M1CM did not affect ALP activity. The enzyme-linked immunosorbent assay detected large amounts of TGF-β1 in M2CM-4 day and M2CM-6 day. The TGF-β blocking antibody suppressed the ALP-enhancing activity of M2CM-4 day (P < .05). Furthermore, the same amount of TGF-β1 as in M2CM-4 day increased ALP activity to a similar level as M2CM-4day-treated HDPCs. CONCLUSIONS: The CM of M2 macrophages enhanced the odontogenic/osteogenic differentiation of HDPCs. M1CM did not affect the ALP activity of HDPCs at least in the absence of M1-type inducers. The effects of M2CM on HDPCs were likely caused by TGF-β1. Therefore, M2 macrophages are expected to support dentin regeneration in dental pulps.
INTRODUCTION: M1 (classically activated) and M2 (alternatively activated) macrophages are known to play primary roles in inflammation and tissue regeneration. To investigate the role of macrophages in dentin regeneration, this study examined the effects of M1 and M2 macrophages on the odontogenic/osteogenic differentiation of human dental pulp cells (HDPCs) using the conditioned media (CM) of the activated human monocyte cell line THP-1. METHODS: M1 and M2 macrophages were induced by lipopolysaccharide/interferon-γ and interleukin-4, respectively, and the phenotypes were confirmed by flow cytometry. Macrophage CM was prepared at 2-day intervals for a period of 6 days, which included the first 2 days of activation. The CM obtained on days 4 (M1CM-4 day and M2CM-4 day) and 6 (M1CM-6 day and M2CM-6 day) were tested for their ability to promote the alkaline phosphatase (ALP) activity of HDPCs. M2CM-4 day was also examined for its effects on the messenger RNA expression of dentin sialophosphoprotein and osteocalcin genes and the matrix mineralization of HDPCs. Tumor necrosis factor alpha and transforming growth factor beta 1 (TGF-β1) in M1CM and M2CM, respectively, were quantified by an enzyme-linked immunosorbent assay. To verify the role of TGF-β1, M2CM-4 day was pretreated by a TGF-β blocking antibody and was examined for its effect on the ALP activity of HDPCs. RESULTS: M2CM-4 day and M2CM-6 day enhanced the ALP activity of HDPCs (P < .05). Furthermore, M2CM-4 day promoted the messenger RNA expression of the dentin sialophosphoprotein gene and matrix mineralization (P < .05), whereas M1CM did not affect ALP activity. The enzyme-linked immunosorbent assay detected large amounts of TGF-β1 in M2CM-4 day and M2CM-6 day. The TGF-β blocking antibody suppressed the ALP-enhancing activity of M2CM-4 day (P < .05). Furthermore, the same amount of TGF-β1 as in M2CM-4 day increased ALP activity to a similar level as M2CM-4day-treated HDPCs. CONCLUSIONS: The CM of M2 macrophages enhanced the odontogenic/osteogenic differentiation of HDPCs. M1CM did not affect the ALP activity of HDPCs at least in the absence of M1-type inducers. The effects of M2CM on HDPCs were likely caused by TGF-β1. Therefore, M2 macrophages are expected to support dentin regeneration in dental pulps.