Marker discrimination of transitional cell stages during osteocytogenesis

Objectives: Since osteocytes derived from osteoblasts, transitional cell stages between osteoblast and osteocyte should be identifiable based on morphological and molecular characters. During the process of osteoblast-to-osteocyte transition, transitional cells are morphologically classified into osteoblastic osteocyte, osteoid osteocyte, young (immature) osteocyte, and old (mature) osteocyte. These osteocytogenesis is accompanied by progressive reduction of several osteoblastic markers and the appearance of osteocytic markers. In this study, we examined the molecular markers to distinguish each stage during the process of osteoblast-to-osteocyte transition. Methods: Tibiae of two and four weeks old rat (Wistar, male) were used for immunohistochemical study with light and fluorescence microscopic observation. Primary antibodies for Runx2, Osterix, DMP-1, GM130, and Cx43 were used. Runx2 and Osterix are used as a marker for osteoblastic differentiation. DMP1 is used as a marker for an extracellular matrix protein expressed chiefly in osteocytes. GM130 is used as a marker for Golgi apparatus. Cx43 is used as a marker for the gap junction protein expressed by functional bone cells. Results: Osteoblast and osteoblastic osteocyte expressed Runx2. Osteoblastic and osteoid osteocytes expressed osterix. However, all of the osteoblastic cells on the bone surface did not always express osterix. Osteoblastic osteocyte which expressed osterix has abundant cytoplasm. Osteoid osteocyte and young osteocyte had DMP1 staining in their Golgi apparatus. Transitional cells from osteoblast to young osteocyte had Cx43 staining at the apical portion of their process. Conclusion: During osteocytogenesis, the osteoblastic and osteoid osteocytes expressed osteoblastic markers (Runx2, Osterix). The osteoid and young osteocytes seemed to accumulate DMP1 in their Golgi apparatus and the old osteocytes did not. These characteristics are used to differentiate each transitional stage from the others. Moreover, the distribution pattern of Cx43 suggested that gap junctional communication occurred in younger osteocyte but not old osteocyte.