In situ proliferation and differentiation of macrophages in dental pulp

The presence of macrophages in dental pulp is well known. However, whether these macrophages proliferate and differentiate in the dental pulp in situ, or whether they constantly migrate from the blood stream into the dental pulp remains unknown. We have examined and compared the development of dental pulp macrophages in an organ culture system with in vivo tooth organs to clarify the developmental mechanism of these macrophages. The first mandibular molar tooth organs from ICR mice aged between 16 days of gestation (E16) to 5 days postnatally were used for in vivo experiments. Those from E16 were cultured for up to 14 days with or without 10% fetal bovine serum. Dental pulp tissues were analyzed with immunohistochemistry to detect the macrophages and with reverse transcription and the polymerase chain reaction (RT-PCR) for the detection of factors related to macrophage development. The growth curves for the in vivo and in vitro cultured cells revealed similar numbers of F4/80-positive macrophages in the dental pulp. RT-PCR analysis indicated the constant expression of myeloid colony-stimulating factor (M-CSF) in both in-vivo- and in-vitro-cultured dental pulp tissues. Anti-M-CSF antibodies significantly inhibited the increase in the number of macrophages in the dental pulp. These results suggest that (1) most of the dental pulp macrophages proliferate and differentiate in the dental pulp without a supply of precursor cells from the blood stream, (2) M-CSF might be a candidate molecule for dental pulp macrophage development, and (3) serum factors might not directly affect the development of macrophages

Immunohistochemical Comparison of Ontogenic Development of Bone Marrow Hematopoiesis in Two Different Ossification Systems

It is well known that the body skeleton is formed by two different types of ossification systems, endochondoral and intramembranous ossification. Bone marrow is the main site of active hematopoiesis after the formation of the bone marrow cavities. However, it is unclear whether the hematopoiesis in the bone marrow of two types of ossification is regulated by the same system or not. In this study, we focused on the ontogenic development of bone marrow hematopoiesis in two different ossification systems using mouse humeral bones and palatal process of maxillary bones. Immunohistochemical and RT-PCR analyses were performed to examine the development of hematopoiesis and the expression of cytokines related to hematopoiesis in the forming bone marrow (16-days gestation stage to 1-day postnatal stage). Immunohistochemical studies showed the sequential difference of hematopoiesis between two different ossification systems. In humeral bone marrow, granulopoiesis appeared first at E16, followed by erythropoiesis from E17. On the contrary, erythropoiesis preceded one day in the maxillary bone marrow at E18, one day before the detection of granulopoiesis. G-SCF and GM-CSF were expressed at every examined stage in both types of bones while M-CSF was not expressed in the humeral bone marrow at E16. Erythropoietin was detetcted in the endothelial cells and its expression was coincident with the onset of erythopoiesis. These results suggest the time kinetic and sequential differences of hematopoiesis in two different ossification systems, which might relate to the differences of hematopoietic microenvironment