Premium
Preparation and characterization of a mouse osteoclast‐like multinucleated cell population
Author(s) -
Akatsu Takuhiko,
Tamura Tatsuya,
Takahashi Naoyuki,
Udagawa Nobuyuki,
Tanaka Sakae,
Sasaki Takahisa,
Yamaguchi Akira,
Nagata Naokazu,
Suda Tatsuo
Publication year - 1992
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650071109
Subject(s) - osteoclast , population , multinucleate , microbiology and biotechnology , tartrate resistant acid phosphatase , bone resorption , bone marrow , acid phosphatase , chemistry , biology , endocrinology , biochemistry , immunology , in vitro , medicine , enzyme , environmental health
We have reported that numerous tartrate‐resistant acid phosphatase‐positive osteoclast‐like multinucleated cells (TRAP + MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1α25‐dihydroxyvitamin D 3 [1α,25‐(OH) 2 D 3 ] (Endocrinology 123 :2600, 1988). In this study, we prepared a TRAP + MNC population using a modified coculture system and examined its osteoclastic properties. TRAP + MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gelcoated dishes. The TRAP + MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP + MNCs was 20,000–40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (∼ 1000 cells per head). The purity of TRAP + MNCs was 5.6 ± 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP + MNCs after density gradient centrifugation was 30–40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [ 125 I]salmon calcitonin (CT) showed abundant CT binding in most TRAP + MNCs. Saturation analysis of [ 125 I]salmon CT indicated a dissociation constant K d for TRAP + MNCs of 8.9 ± 0.7 × 10 10 M and 16.5 ± 1.5 ± 10 6 binding sites per cell. These results were similar to the value (3.5 × 10 −10 M) and the number of binding sites (3.3 × 10 6 per cell) in isolated rat OCLs. Displacement curves for [ 125 I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: salmon CT at 10 −10 M, human CT at 10 −8 M; ED 50 : salmon CT, 2.2 × 10 −11 M, human CT, 1.3 × 10 −9 M) in the MNC preparation. These results indicate that a large number of mouse TRAP + MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.