gms | German Medical Science

Objectives: Compromised bone repair is often observed in osteoporotic patients. Beyond the negative influence of estrogen-deficiency on bone mass, pathomechanisms are poorly understood. Previously, we found increased levels of the estrogen-responsive and pro-inflammatory cytokine Midkine (Mdk) and Interleukine-6 (IL-6) in the serum and fracture hematoma of ovariectomized (OVX) mice, a model for postmenopausal osteoporosis. This was associated with increased neutrophil infiltration in the hematoma. These data indicate that an increased immune response might contribute to disturbed bone regeneration in estrogen-depleted subjects. To prove clinical relevance we here investigated the early immune response after fracture in male and postmenopausal female fracture patients by measuring Mdk and IL-6 levels. Furthermore, we investigated if sera of postmenopausal patients negatively affect osteogenic differentiation in vitro.

Methods: In 23 patients with isolated long bone fracture, serum Mdk and IL-6 levels were measured at the day of surgery (d0), d14 and d42 after surgery. Subgroup-analysis between men (n=6) and postmenopausal women (n=16) was conducted at d0. 26 healthy donors served as controls. In vitro, human mesenchymal stem cells (MSC) underwent osteogenic differentiation for 10 days. MSCs were cultured with 10% serum from controls, male, and postmenopausal fracture patients (n=6-10). Half of the cells were treated with an inhibitory Mdk-antibody (Mdk-Ab). Osteogenic differentiation was evaluated by gene expression analysis (Gapdh, Alpl, Bglap) and AP staining. Statistics: Student's t-test/Kruskal-Wallis test, p≤0.05.

Results and conclusion: In fracture patients, Mdk and IL-6 serum levels were significantly increased compared to healthy controls at all time points. Subgroup analysis revealed significantly higher serum levels of Mdk (1009±555 vs. 511±311 pg/ml) but not of IL-6 in postmenopausal patients compared to males. In vitro analysis showed that MSCs cultured with sera from male and female fracture patients displayed no differences in Alpl expression, but significantly reduced Bglap expression compared to healthy controls. MSCs cultured with sera from postmenopausal patients displayed reduced Alpl and Bglap expression compared to males (Alpl: males 4.24±3.54 females 2.41±1.26; Bglap: males 0.162±0.06 females 0.123±0.01). Treatment with Mdk-Ab increased Alpl and Bglap expression in MSC cultured with female patient sera (p≤0.05), while this effect was not detected in males. AP staining confirmed these results.

Postmenopausal fracture patients displayed increased Mdk levels after fracture, thus confirming our previous experimental data in mice. Because Mdk is a negative regulator of bone formation, this might contribute to impaired fracture healing. Confirming, our in vitro data showed increased osteogenic differentiation of human MSCs treated with serum of postmenopausal patients after Mdk-Ab treatment, indicating that Mdk might be a target to improve osteoporotic bone repair in patients.