Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1α,25‐dihydroxyvitamin D₃ have ruffled borders and resorb dentine

Abstract

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1 alpha,25(OH)2D3 were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that 1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1 alpha,25(OH)2D3 exhibit non-functional basic features of osteoclast morphology, and 2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.

Histological Disorders Related to the Focal Disappearance of the Epiphyseal Growth Plate in Rats Induced by High Dose of Vitamin A.

“…Therefore, the cartilage matrix with the deposition of type I collagen may induce the invasion of osteoclasts to the uncalcified cartilage matrix in VA rats on Day 12. However, it was unclear whether these osteoclasts absorbed the uncalcified cartilage matrix, because induction of osteoclastic activity has been reported to require the adhesion of osteoclasts to the calcified matrix [15]. In the epiphyseal growth plate in VA rats on Day 12, osteoblasts and osteoid were detected on the surfaces of the eroded areas in accompany with osteoclasts.…”

The Role of the Plasminogen System in Bone Resorption In Vitro

“…(24) Cocultures were treated with PGE 2 (2 × 10 −6 M) and the cells were recovered after 6 days by collagenase (2 mg/ml) treatment. Cells were suspended in ␣-MEM with 10% FCS, counted and plated on dentine slices (4 mm, thickness 200 m, prepared as described (25) ) in a 96-well culture plate. After 2 h, the dentine slices were washed carefully with ␣-MEM, transferred to a 24-well culture plate, and cultured for an additional 48 h in ␣-MEM with 0.1% BSA.…”

Recent advances in the ultrastructural assessment of osteoclastic resorptive functions

“…The majority of pale vesicles and vacuoles do not contain such crystal fragments. Some osteoclastic lysosomes, of variable electron density, do, however, have observable crystal fragments (Sasaki et al, 1989). This indicates that most bone crystals are demineralized at an extracellular site beneath the osteoclastic ruff led border.…”