Suppression of TNF-α activity by immobilization rescues Mkx expression and attenuates tendon ossification in a mouse Achilles tenotomy model

Traumatic heterotopic ossification is a condition in which extraskeletal bone formation occurs in soft tissues after injury. It most commonly occurs in patients who had major orthopedic surgery and in those with severe extremity injuries. The lesion causes local pain and can impair motor function of the affected limb, but there is currently no established prophylaxis or treatment for this condition. In this study, we show that immobilization at an early stage of the inflammatory response after injury can attenuate ossification formation in a murine Achilles tenotomy model. Gene expression analysis revealed a decrease in the expression of Tnf and an increase in the expression of Mkx, which encodes one of the master regulators of tendon differentiation, Mohawk. Notably, we found that TNF-alpha suppressed the expression of Mkx transcripts and accelerated the osteogenic differentiation of tendon-derived mesenchymal stem cells (MSCs), suggesting that TNF-alpha acts as a negative regulator of Mkx transcription. Consistent with these findings, pharmaceutical inhibition of TNF-alpha increased the expression of Mkx transcripts and suppressed bone formation in this mouse model. These findings reveal the previously unrecognized involvement of TNF-alpha in regulating tendon MSC fate through suppression of Mkx expression and suggest that TNF-alpha is a potential target for preventing traumatic heterotopic ossification.