Effect of Tumor Necrosis Factor to Chondrocytes in Endochondral Ossification

2001 Fiscal Year Final Research Report Summary

• Project/Area Number: 12671391
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Orthopaedic surgery
• Research Institution: Tohoku University
• Principal Investigator: AIZAWA Toshimi Tohoku University School of Medicine, Dept. of Orthopaedic Surgery, Assistant Professor, 大学院・医学研究系体性外科学, 助手 (50282132)
• Co-Investigator(Kenkyū-buntansha): OONUMA Masahiro Tohoku University School of Medicine, Dept. of Orthopaedic Surgery, Medical Staff, 医学部・附属病院, 医員 ; KAWAMATA Tomomaro Tohoku University School of Medicine, Dept. of Orthopaedic Surgery, Assistant Professor, 医学部・附属病院, 助手 (70333804)
• Project Period (FY): 2000 – 2001
• Keywords: fracture healing / endochondral ossification / apotosis / chondrocyte / tumor necrosis factor

Research Abstract

In endochondral ossification, chondrocytes change their morphology and functions and are ultimately removed by the process of programmed cell death (apoptosis). A variety of apoptosis inducing signals and death factors have been characterized including tumor necrosis factor-alpha (TNF-α), Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL). However, there is little known regarding the activity of these factors in the process of fracture healing. Using RNase protection assay (RPA) and immunohistochemistry, this study sought to determine if these factors might participate in endochondral fracture healing. A closed, transverse fracture was made in the right tibiae of 8-10 week old BALB/C mice. Mice were euthanized at 1, 2 and 3 weeks post-fracture. The tibiae were harvested and studied for the expression of caspase-8, a key enzyme of apoptosis. TNF-α, FasL and TRAIL, were assayed using RPA for mRNA and immunostaining for protein. At each time point, caspase-8, FasL and its receptor Fas receptor (FasR), TNF-α and its receptor p55, and TRAIL were detected by RPA. Immunostainings clearly showed that caspase-8, TNF-α, FasL, FasR and TRAIL were expressed in fracture callus chondrocytes. Additionally, the mice in which TNF-α receptors were ablated showed very little expression of caspase-8 by RPA. These findings suggest that cartilaginous callus is replaced by new bone during endochondral fracture healing and chondrocyte removal by apoptosis is controlled by several death factors via autocrine and paracrine mechanisms, particularly TNF-α might play a major role in chondrocyte death.