Immunohistochemical Study of Apoptosis of Osteoclasts During Bone Remodeling

Objectives: It is well reported that mononuclear cells under physiological conditions die by apoptosis. But the apoptosis of multinucleated cells, osteoclasts, during bone remodeling remains unclear. This study was designed to establish the apoptosis of osteoclasts on alveolar bone during bone remodeling.

Methods: Ten, 1-week-old male Wistar strain rats were used in this study. Mandibles were fixed in 4% formaldehyde for 24 hrs at 4°C, decalcified in 5% EDTA, and embedded in paraffin. The specimens were sequentially cut into 5µm sections. Apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) (ApopTag Peroxdase In Situ Apoptosis Detection Kit; Chemicon, USA), and then tartrate-resistant acid phosphatase (TRAP) activity was determined on the same sections. The sections were observed by light microscopy.

Results: Osteoclasts were TRAP-positive, and they were multinucleated cells forming resorptive lacunae under brush borders on alveolar bone. The nuclei of TRAP-positive osteoclasts performing bone resorption were generally TUNEL-negative. Around these osteoclasts, TRAP-positive structures with one or two TUNEL-positive nuclei, and TRAP-positive structures without any nuclei were often observed. TRAP-positive fragments were also seen in fibroblastic cells and around blood vessels.

Conclusion: The presence of TRAP-positive structures with TUNEL-positive nuclei shows the apoptosis of osteoclasts during bone remodeling. The structures shown in the results suggest that osteoclasts undergoing apoptosis fragment into various sized cellular parts including two or fewer nuclei, and that these fragments are phagocytosed by fibroblastic cells and/or migrate into blood vessels.