Estrogen Deficiency Exacerbates Traumatic Heterotopic Ossification in Mice

Background: Traumatic heterotopic ossification (HO) is a devastating sequela of orthopedic surgeries and traumatic injuries; however, few studies have explored the effects of the estrogen-deficient state on HO formation. In the present study, we investigated the impact of estrogen deficiency on ectopic cartilage and bone formation in tendon after Achilles tenotomy in an ovariectomized mouse model. Methods: A total of 45 female C57BL/6 mice were randomly divided into three groups: sham-operated (control), estrogen depletion by ovariectomy (OVX) and OVX with 17(3-estradiol supplementation (OVX + E2), 2 ), with 15 animals in each group. Three weeks after OVX, all mice were subjected to an Achilles tenotomy using a posterior midpoint approach to induce HO. At 1, 3 and 9 weeks after tenotomy, the left hind limbs were harvested for histology, immunohistochemistry and immunofluorescence evaluations. The volume of ectopic bone was assessed by micro-CT. Results: Mice in the OVX group formed more ectopic cartilage 3 weeks after tenotomy, as well as ectopic bone 9 weeks after tenotomy, compared to the control group. Estrogen deficiency resulted in more severe inflammatory infiltration at the injury sites 1 week after tenotomy, involving the recruitment of more macrophages and mast cells, as well as increasing the expressions of pro- inflammatory mediators, including IL-1(3, IL-6, and TNF-alpha. Moreover, the local TGF-(3/SMAD signaling pathway was dysregulated after OVX, which manifested as upregulated expressions of TGF-(3 and pSMAD2/3. E2 2 supplementation protected against OVXinduced HO deterioration, inhibited inflammatory infiltration, and downregulated the TGF-(3/SMAD signaling pathway. Conclusion: Estrogen deficiency exacerbated HO formation in the Achilles tenotomy model. These findings might be attributable to the disturbance of the inflammatory response and the activation of TGF-(3/SMAD signaling at the injury sites during the early stages of HO development.