29. Establishment and Characterization of Tartrate-resistant Acid Phosphatase (TRAP) Positive Cell Clones from Hamster Bone Marrow: 1. Cartilage Degrading Activity

Hisako Sakiyama, Koichi Nakagawa, Riako Masuda, Kazuko Kuriiwa, Modori Honjo, Yuki Takada and Kazuko Yoshida

Keywords: TRAP, clone, morphology, cartilage, degradation

Long bones develop from cartilage, devoid of blood and lymph capillaries, and consisting of amorphous cartilage matrix and chondrocytes. Chondrocytes are programmed to die after differentiating from resting, proliferating, maturing and hypertrophic cells, and the cartilage is replaced by bone marrow from where endochondral ossification starts. The calcified cartilage matrix decreases in parallel with chondrocyte hypertrophic differentiation. Degenerative enzymes such as MMP-9 and complement C1s are secreted during this process. After the programmed cell death of hypertrophic chondrocytes, the remaining sepia of the cartilage matrix is resorbed by cells such as septoclasts. Tartrate-resistant acid phosphatase (TRAP) positive cells which are considered to play a role in the remodeling are often observed at epiphyseal/metaphyseal and metaphyseal/diaphyseal borders. TRAP is used as a marker enzyme for chondroclast/ osteoclast differentiation, the function of which is considered to relate to cartilage/bone resorption. TRAP dephosphorylates bone phosphoproteins, and mice lacking TRAP have been shown to be delayed in cartilage mineralization and defective in osteoclastic bone turnover. However, cartilage resorbing TRAP positive cells (chondroclasts) and bone resorbing TRAP positive cells (osteoclasts) have not been proven to be different cells.

Osteoclast differentiation has been studied by a model system, the coculture of mouse stromal cells and bone marrow cells. Using this system, Chen and Li have immortalized osteoclastogenic cells with SV4OT antigen. Four out of 72-isolated cell lines were induced to generate TRAP positive osteoclast precursor cells when cocultured with stromal cells in the presence of 1, 25-(OH)₂D₃, but none became TRAP positive when cultured alone. From p53-/-mouse bone marrow cells, Miyamoto et al. established a macrophage-like cell line which differentiated to osteoclasts in the presence of stromal sells and l, 25-(OH)₂D₃.

In our laboratory, coculture of hamster bone marrow cells with irradiated hamster chondrocytes and establishment of TRAP positive clones were carriedout. In this system, 1, 25-(OH)₂D₃, a stimulator of TRAP positive cell formation, was not required. Growth characterization and chondrolytic activities of these clones were examined. The isolated clones were designated as CCP-1 through CCP-16. CCP-1, CCP-2, CCP-6 and CCP-7 were mainly used in this study. Morphologically, the clones resembled macrophages, having numerous vacuoles in the cytoplasm, and filopodia and microvilli on the cell surface. The clones fused to form multinuclear giant cells. Their growth was stimulated by chondrocyte-conditioned medium and human M-CSF. The clones were cultured on hamster epiphyseal or human articular cartilage in order to investigate their physiological functions. All tested clones were observed to degrade and invade the cartilage matrix, and also deposit calcified materials in the matrix. However, electron microscopic examinations showed that the cells did not from a ruffled border or clear zone, both of which are characteristics of osteoclasts.

To date, the clones still remain TRAP positive after four years maintenance in the absence of stromal chondrocytes and 1, 25-(OH)₂D₃.

Publications:
1) Sakiyama, H., Kaji K., Nakagawa, K. and Nagino, K. : Cell Biochem. Func. 16, 159-163, 1998.
2) Inoue, N., Saito, T., Masuda, R., Suzuki, Y., Ohtomi, M. and Sakiyama, H.: Hum. Genet. 103, 415-418, 1998.
3) Nakagawa, K, Sakiyama, H., Tuchida, T., Yamaguchi, K., Toyoguchi, T., Masuda, R. and Moriya, H.: Ann. Rheum. Dis. 58, 175-181, 1999.
4) Suzuki, G., Sawa, H., Kobayashi, Y., Nakagawa, K., Uzawa, A., Sakiyama, H., Kakinuma, H., Iwabuchi, K. and Nagashima, K.: J. Immunol. 162, 5981-5985, 1999.
5) Takada, Y., Sakiyama, H., Kuriiwa, K., Masuda, R., Inoue, N., Nakagawa, K., Itano, N., Saito, T., Yamada, T. and Kimata, K.: Cell Tissue Res., in press.