gms | German Medical Science

Objectives: Clinical evidence suggests that fracture healing is impaired in postmenopausal, osteoporotic women (Cheung et al. 2016). Confirming, experimental studies demonstrated that ovariectomized animals displayed impaired callus formation during fracture healing (Wang et al. 2005; Beil et al. 2010). It is known that estrogen-deficiency affects the immune system and the inflammatory response during wound healing (Pacifici, 2008; Routley et al. 2009). Since a balanced immune response is required for proper fracture healing (Claes et al. 2012), we were interested if the early immune response after fracture is affected by estrogen-deficiency.

Methods: 3-4 month old female C57BL/6J mice (n=32) were bilaterally ovariectomized (OVX) or sham-operated. Eight weeks after OVX, mice received a femur osteotomy stabilized by an external fixator. The effects of estrogen depletion on the presence of immune cells (neutrophils, monocytes, macrophages, B- and T-lymphocytes) and pro-inflammatory cytokines (midkine (Mdk), interleukin-6 (IL-6), CCL2, CXCL1) were evaluated by flow cytometry or immunohistochemistry on day 1 and 3 after fracture. Statistics: Shapiro-Wilk normality test, Student's t-test or ANOVA; p<0.05.

Results and Conclusion: On day 1 after fracture, FACS analysis revealed no significant differences in immune cell populations between sham- and OVX-mice, suggesting that the initial recruitment of immune cells to the fracture callus was not affected by estrogen-deficiency. However, on day 3 after fracture, immunohistochemical staining revealed that OVX-mice displayed significantly higher numbers of neutrophils (+220%) in the periostal callus compared to sham-mice, indicating a prolonged recruitment or increased survival of neutrophils in the absence of estrogen. Furthermore, the local expression of the estrogen-responsive and pro-inflammatory cytokine Mdk and IL-6 expression were increased in OVX-mice on day 3 after fracture, implying that both molecules may be involved in the increased presence of neutrophils. Confirming, antagonizing Mdk by Mdk-Ab treatment directly after fracture decreased the number of neutrophils (-58%) in the fracture callus and local IL-6 expression in OVX-mice.

These data indicate that estrogen-deficiency may influence the early inflammatory phase after fracture by increasing the number of neutrophils and the presence of inflammatory cytokines in the fracture hematoma. Because neutrophil numbers and local IL-6 expression were decreased after Mdk-Ab treatment, we hypothesize that Mdk and IL-6 may interact in the estrogen effects on neutrophil recruitment or survival. The increased inflammatory response after fracture could possibly contribute to delayed healing after estrogen-deficiency.