The inflammatory phase of fracture healing is influenced by oestrogen status in mice

Abstract Background Fracture healing is known to be delayed in postmenopausal, osteoporotic females under oestrogen-deficient conditions. Confirming this, experimental studies demonstrated impaired callus formation in ovariectomised animals. Oestrogen-deficiency is known to affect the immune system and the inflammatory response during wound healing. Because a balanced immune response is required for proper bone healing, we were interested to ascertain whether the early immune response after facture is affected by oestrogen depletion. Methods To address the above question, female mice received either a bilateral ovariectomy (OVX) or were sham-operated, and femur osteotomy was performed 8 weeks after OVX/sham operation. The effects of OVX on the presence of immune cells and pro-inflammatory cytokines were evaluated by flow cytometry and immunohistochemistry of the fracture calli on days 1 and 3 after fracture. Results One day after fracture, immune cell numbers and populations in the fracture haematoma did not differ between OVX- and sham-mice. However, on day 3 after fracture, OVX-mice displayed significantly greater numbers of neutrophils. Local expression of the oestrogen-responsive and pro-inflammatory cytokine midkine (Mdk) and interleukin-6 (IL-6) expression in the fracture callus were increased in OVX-mice on day 3 after fracture compared with sham-mice, indicating that both factors might be involved in the increased presence of neutrophils. Confirming this, Mdk-antibody treatment decreased the number of neutrophils in the fracture callus and reduced local IL-6 expression in OVX-mice. Conclusions These data indicate that oestrogen-deficiency influences the early inflammatory phase after fracture. This may contribute to delayed fracture healing after oestrogen depletion

Inhibition of Midkine Augments Osteoporotic Fracture Healing.

The heparin-binding growth and differentiation factor midkine (Mdk) is proposed to negatively regulate osteoblast activity and bone formation in the adult skeleton. As Mdk-deficient mice were protected from ovariectomy (OVX)-induced bone loss, this factor may also play a role in the pathogenesis of postmenopausal osteoporosis. We have previously demonstrated that Mdk negatively influences bone regeneration during fracture healing. Here, we investigated whether the inhibition of Mdk using an Mdk-antibody (Mdk-Ab) improves compromised bone healing in osteoporotic OVX-mice. Using a standardized femur osteotomy model, we demonstrated that Mdk serum levels were significantly enhanced after fracture in both non-OVX and OVX-mice, however, the increase was considerably greater in osteoporotic mice. Systemic treatment with the Mdk-Ab significantly improved bone healing in osteoporotic mice by increasing bone formation in the fracture callus. On the molecular level, we demonstrated that the OVX-induced reduction of the osteoanabolic beta-catenin signaling in the bony callus was abolished by Mdk-Ab treatment. Furthermore, the injection of the Mdk-Ab increased trabecular bone mass in the skeleton of the osteoporotic mice. These results implicate that antagonizing Mdk may be useful for the therapy of osteoporosis and osteoporotic fracture-healing complications

Chronic psychosocial stress compromises the immune response and endochondral ossification during bone fracture healing via β-AR signaling.

Chronic psychosocial stress/trauma represents an increasing burden in our modern society and a risk factor for the development of mental disorders, including posttraumatic stress disorder (PTSD). PTSD, in turn, is highly comorbid with a plethora of inflammatory disorders and has been associated with increased bone fracture risk. Since a balanced inflammatory response after fracture is crucial for successful bone healing, we hypothesize that stress/trauma alters the inflammatory response after fracture and, consequently, compromises fracture healing. Here we show, employing the chronic subordinate colony housing (CSC) paradigm as a clinically relevant mouse model for PTSD, that mice subjected to CSC displayed increased numbers of neutrophils in the early fracture hematoma, whereas T lymphocytes and markers for cartilage-to-bone transition and angiogenesis were reduced. At late stages of fracture healing, CSC mice were characterized by decreased bending stiffness and bony bridging of the fracture callus. Strikingly, a single systemic administration of the β-adrenoreceptor (AR) blocker propranolol before femur osteotomy prevented bone marrow mobilization of neutrophils and invasion of neutrophils into the fracture hematoma, both seen in the early phase after fracture, as well as a compromised fracture healing in CSC mice. We conclude that chronic psychosocial stress leads to an imbalanced immune response after fracture via β-AR signaling, accompanied by disturbed fracture healing. These findings offer possibilities for clinical translation in patients suffering from PTSD and fracture

Midkine-antibody (Mdk-Ab) treatment increased the bone content in the intact femur of ovariectomized (OVX) mice after short-time treatment.

<p>Micro-computed tomography (μCT) analysis of the cortical bone at the midshaft of the intact femur volume of interest 2 (VOI 2): A) cortical tissue mineral density (TMD) and B) cortical thickness. μCT analysis of the distal part of the intact femur (VOI 3): C) trabecular TMD, D) bone volume to tissue volume ratio, E) trabecular thickness and F) trabecular number. *Significantly different from sham+vehicle or OVX+vehicle group (p<0.05) by Kruskal-Wallis test. (n = 6–7 per group.) G) Three-dimensional reconstructions of the trabecular region of the distal intact femur (VOI 3), representative images are shown.</p

Midkine-antibody (Mdk-Ab) treatment increased the bone content in vertebral bodies of ovariectomized (OVX) mice after short-time treatment.

<p>Micro-computed tomography (μCT) analysis of the vertebral bodies (volume of interest 4): A) two-dimensional images of the second caudal vertebral body, representative images are shown. B) Trabecular tissue mineral density, C) trabecular bone volume to tissue volume ratio, D) trabecular thickness and E) trabecular number. *Significantly different from sham+vehicle or OVX+vehicle group (p<0.05) by Kruskal-Wallis test. (n = 6−7 per group).</p

Midkine-antibody (Mdk-Ab) treatment accelerated osteoporotic fracture healing.

<p>Biomechanical, micro-computed tomography (μCT) and histomorphometric analysis of the fractured femurs at day 23. Biomechanical testing: A) relative flexural rigidity of the fractured femur in comparison with intact femur determined by biomechanical testing. Parameters determined by μCT analysis (volume of interest 2): B) bone volume to tissue volume ratio and C) tissue volume (n = 6–7 per group). Parameters determined by histomorphometric analysis of the whole fracture callus at day 10 (white bars) and day 23 (grey bars): D) bone area to tissue area ratio, E) cartilage area to tissue area ratio and F) fibrous tissue area to tissue area ratio. *Significantly different from sham+vehicle or OVX+vehicle group (p<0.05) by Kruskal-Wallis test. (n = 5–7 per group.) G) Representative images of sections from undecalcified femurs at day 23, stained using Giemsa; scale bar: 250 μm.</p

Midkine (Mdk) serum levels during fracture healing in 3-month-old sham-operated and ovariectomized (OVX) mice in pg/ml.

<p>Midkine (Mdk) serum levels during fracture healing in 3-month-old sham-operated and ovariectomized (OVX) mice in pg/ml.</p

Cellular parameters in the fracture callus and the intact femur at day 23.

<p>Cellular parameters in the fracture callus and the intact femur at day 23.</p