TNFα-dependent mTOR activity is required for tenotomy-induced ectopic ossification in mice

Introduction Ectopic ossifications often occur in skeletal muscles or tendons following local trauma or internal hemorrhage, and occasionally cause severe pain that limits activities of daily living. However, mechanisms underlying their development remain unknown. Materials and methods The right Achilles tendon in 8-week-old female or male mice was dissected. Some mice were injected intraperitoneally either with phosphate-buffered saline, dimethyl sulfoxide, cimetidine, rapamycin, celecoxib or loxoprofen for 10 weeks. One week after surgery, immunohistochemical analysis was performed for mTOR, TNF alpha or F4/80. Ten weeks after surgery, ectopic ossification at the tenotomy site was detected by 3D micro-CT. Results Ectopic ossification was seen at dissection sites in all wild-type mice by dissection of the Achilles tendon. mTOR activation was detected at dissection sites, and development of ectopic ossification was significantly inhibited by administration of rapamycin, an mTOR inhibitor, to wild-type mice. Moreover, administration of the histamine 2 blocker cimetidine, which reportedly inhibits ectopic ossification in tendons, was not effective in inhibiting ectopic ossification in our models. TNF alpha-expressing F4/80-positive macrophages accumulate at dissection sites and that ectopic ossification of the Achilles tendon dissection was significantly inhibited in TNF alpha-deficient mice in vivo. Ectopic ossification is significantly inhibited by administration of either celecoxib or loxoprofen, both anti-inflammatory agents, in wild-type mice. mTOR activation by Achilles tendon tenotomy is inhibited in TNFa-deficient mice. Conclusion The TNF alpha-mTOR axis could be targeted therapeutically to prevent trauma-induced ectopic ossification in tendons.