Melanie Haffner-Luntzer

The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 g peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (−81%) and bone formation (−80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations.

The Wnt Serpentine Receptor Frizzled-9 Regulates New Bone Formation in Fracture Healing

Wnt signaling is a key regulator of bone metabolism and fracture healing. The canonical Wnt/β-catenin pathway is regarded as the dominant mechanism, and targeting this pathway has emerged as a promising strategy for the treatment of osteoporosis and poorly healing fractures. In contrast, little is known about the role of non-canonical Wnt signaling in bone. Recently, it was demonstrated that the serpentine receptor Fzd9, a Wnt receptor of the Frizzled family, is essential for osteoblast function and positively regulates bone remodeling via the non-canonical Wnt pathway without involving β-catenin-dependent signaling. Here we investigated whether the Fzd9 receptor is essential for fracture healing using a femur osteotomy model in Fzd9 −/− mice. After 10, 24 and 32 days the fracture calli were analyzed using biomechanical testing, histomorphometry, immunohistochemistry, and micro-computed tomography. Our results demonstrated significantly reduced amounts of newly formed bone at all investigated healing time points in the absence of Fzd9 and, accordingly, a decreased mechanical competence of the callus tissue in the late phase of fracture healing. In contrast, cartilage formation and numbers of osteoclasts degrading mineralized matrix were unaltered. β-Catenin immunolocalization showed that canonical Wnt-signaling was not affected in the absence of Fzd9 in osteoblasts as well as in proliferating and mature chondrocytes within the fracture callus. The expression of established differentiation markers was not altered in the absence of Fzd9, whereas chemokines Ccl2 and Cxcl5 seemed to be reduced. Collectively, our results suggest that non-canonical signaling via the Fzd9 receptor positively regulates intramembranous and endochondral bone formation during fracture healing, whereas it does not participate in the formation of cartilage or in the osteoclastic degradation of mineralized matrix. The finding that Fzd9, in addition to its role in physiological bone remodeling, regulates bone repair may have implications for the development of treatments for poorly or non-healing fractures.

Midkine-Deficiency Delays Chondrogenesis during the Early Phase of Fracture Healing in Mice

The growth and differentiation factor midkine (Mdk) plays an important role in bone development and remodeling. Mdk-deficient mice display a high bone mass phenotype when aged 12 and 18 months. Furthermore, Mdk has been identified as a negative regulator of mechanically induced bone formation and it induces pro-chondrogenic, pro-angiogenic and pro-inflammatory effects. Together with the finding that Mdk is expressed in chondrocytes during fracture healing, we hypothesized that Mdk could play a complex role in endochondral ossification during the bone healing process. Femoral osteotomies stabilized using an external fixator were created in wildtype and Mdk-deficient mice. Fracture healing was evaluated 4, 10, 21 and 28 days after surgery using 3-point-bending, micro-computed tomography, histology and immunohistology. We demonstrated that Mdk-deficient mice displayed delayed chondrogenesis during the early phase of fracture healing as well as significantly decreased flexural rigidity and moment of inertia of the fracture callus 21 days after fracture. Mdk-deficiency diminished beta-catenin expression in chondrocytes and delayed presence of macrophages during early fracture healing. We also investigated the impact of Mdk knockdown using siRNA on ATDC5 chondroprogenitor cells in vitro. Knockdown of Mdk expression resulted in a decrease of beta-catenin and chondrogenic differentiation-related matrix proteins, suggesting that delayed chondrogenesis during fracture healing in Mdk-deficient mice may be due to a cell-autonomous mechanism involving reduced beta-catenin signaling. Our results demonstrated that Mdk plays a crucial role in the early inflammation phase and during the development of cartilaginous callus in the fracture healing process.

Systemic treatment with the sphingosine‐1‐phosphate analog FTY720 does not improve fracture healing in mice

Sphingosine‐1‐phosphate (S1P) has recently been recognized as a crucial coupling molecule of osteoclast and osteoblast activity provoking osteoanabolic effects. Targeting S1P receptors could, therefore, be a potential strategy to support bone formation in osteopenic diseases or in fracture repair. Here we investigated whether systemic treatment with the S1P analog FTY720 (Fingolimod) could improve fracture healing. Twelve‐week‐old, female C57BL/6 mice received an osteotomy of the femur, which was stabilized using an external fixator. The mice received a daily subcutaneous injection of either FTY720 (6 mg/kg) or vehicle from the third postoperative day. Fracture healing was evaluated after 10 and 21 days using biomechanical testing, µ‐computed tomography, and histomorphometry. Because FTY720 is supposed to influence osteoclast recruitment, osteoclasts were identified in the fracture callus by staining for tartrate resistant acid phosphatase (TRAP). There were no significant differences in callus mechanical properties, tissue composition and osteoclast number between the groups, suggesting that systemically applied FTY720 did not influence bone regeneration in this model of regular fracture healing. Even if further studies should test the potency of FTY720 under unfavorable healing conditions, we conclude that the effect of systemically applied FTY720 on fracture healing might be inferior compared to other anabolic treatments.

Distinct Effects of IL-6 Classic and Trans-Signaling in Bone Fracture Healing

Bone healing is a complex process with closely linked phases of inflammation, regeneration, and remodeling. IL-6 may crucially regulate this process; however, the underlying mechanisms are unclear. IL-6 signals are transmitted via the transmembrane glycoprotein 130 by two distinct mechanisms: classic signaling using the membrane-anchored IL-6 receptor and trans-signaling using its soluble form. Herein, we investigated the hypothesis that IL-6 classic and trans-signaling have different functions during bone healing. To investigate fracture healing, 12-week-old C57BL/6J mice underwent a femur osteotomy. To study the function of IL-6 during the inflammatory phase, either an anti-IL-6 antibody, which inhibits IL-6 classic and trans-signaling, or soluble glycoprotein 130 fusion protein, which selectively blocks trans-signaling, was injected after 30 minutes and 48 hours. To analyze IL-6 effects in the repair phase, compounds were injected from day 7 onwards. Global IL-6 inhibition in the early phase after fracture reduced systemic inflammation, the recruitment of immune cells, and bone regeneration, resulting in delayed fracture healing. Global IL-6 inhibition during the repair phase disturbed bone formation and remodeling. In contrast, inhibition of IL-6 trans-signaling exerted minor effects on the immune response and did not influence bone repair, suggesting that the classic pathway accounts for most of the effects observed after global IL-6 inhibition. Our results reveal that IL-6 classic signaling, but not IL-6 trans-signaling, is essential for bone repair.

Loss of p53 compensates osteopenia in murine Mysm1 deficiency

Histone modifications critically contribute to the epigenetic orchestration of bone homeostasis—in part, by modifying the access of transcription factors to specific genes involved in the osteogenic differentiation process of bone marrow mesenchymal stem cells (MSCs) and osteoblasts. Based on our previous finding that histone H2A deubiquitinase 2A‐DUB/Mysm1 interacts with the p53 axis in hematopoiesis and tissue development, we analyzed the molecular basis of the skeletal phenotype of Mysm1‐deficient mice and dissected the underlying p53‐dependent and ‐independent mechanisms. Visible morphologic, skeletal deformations of young Mysm1‐deficient mice— including a kinked and truncated tail and shortened long bones—were associated with osteopenia of long bones. On the cellular level, Mysm1‐deficient primary osteoblasts displayed reduced potential to differentiate into mature osteoblasts, as indicated by decreased expression of osteogenic markers. Reduced osteogenic differentiation capacity of Mysm1‐deficient osteoblasts was accompanied by an impaired induction of osteogenic transcription factor Runx2. Osteogenic differentiation of Mysm1−/− MSCs, however, was not compromised in vitro. In line with defective hematopoietic development of Mysm1‐deficient mice, Mysm1−/− osteoclasts had reduced resorption activity and were more prone to apoptosis in TUNEL assays. Skeletal alterations and osteopenia of Mysm1‐deficient mice were phenotypically completely rescued by simultaneous ablation of p53 in p53−/−Mysm1−/− double‐deficient mice— although p53 deficiency did not restore Runx2 expression in Mysm1−/− osteoblasts on the molecular level but, instead, enhanced proliferation and osteogenic differentiation of MSCs. In summary, our results demonstrate novel roles for Mysm1 in osteoblast differentiation and osteoclast formation, resulting in osteopenia in Mysm1‐deficient mice that could be abrogated by the loss of p53 from increased osteogenic differentiation of Mysm1−/−p53−/− MSCs.— Haffner‐Luntzer, M., Kovtun, A., Fischer, V., Prystaz, K., Hainzl, A., Kroeger, C. M., Krikki, I., Brinker, T. J., Ignatius, A., Gatzka, M. Loss of p53 compensates osteopenia in murine Mysml deficiency. FASEB J. 32, 1957–1968 (2018). www.fasebj.org

Calcium and vitamin-D deficiency marginally impairs fracture healing but aggravates posttraumatic bone loss in osteoporotic mice

Calcium and vitamin-D (Ca/VitD) deficiency is a major risk factor for osteoporosis. It may also contribute to the compromised bone healing frequently observed in osteoporotic patients, since calcium is essential for fracture-callus mineralization. Additionally, clinical data suggest systemic bone loss following fracture, which may aggravate osteoporosis and thus increase the risk for fragility fractures in osteoporotic patients further. However, the role of Ca/VitD in fracture healing and posttraumatic bone turnover has to date been poorly investigated. Here, we studied bone regeneration and posttraumatic bone turnover in C57BL/6 J mice with ovariectomy-induced osteoporosis. Mice were fed a standard or a Ca/VitD-deficient diet. Notably, fracture healing was only marginally disturbed in Ca/VitD-deficient mice. However, deficient mice displayed significantly increased serum parathyroid hormone levels and osteoclast activity, as well as reduced bone mass in the intact skeleton post-fracture, suggesting considerably enhanced calcium mobilization from the intact skeleton during bone regeneration. Ca/VitD supplementation initiated post-fracture prevented posttraumatic bone loss by reducing bone resorption and furthermore improved bone repair. These results imply that adequate Ca/VitD supply post-fracture is essential to provide sufficient calcium for callus-mineralization in order to prevent posttraumatic bone loss and to reduce the risk for secondary fractures in osteoporotic patients with Ca/VitD deficiency.

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.

Antagonizing Midkine Accelerates Fracture Healing in Mice by Enhanced Bone Formation in the Fracture Callus

Previous findings suggest that the growth and differentiation factor midkine (Mdk) is a negative regulator of osteoblast activity and bone formation, thereby raising the possibility that a specific Mdk antagonist might improve bone formation during fracture healing.

Chronic psychosocial stress disturbs long-bone growth in adolescent mice

ABSTRACT Although a strong association between psychiatric and somatic disorders is generally accepted, little is known regarding the interrelationship between mental and skeletal health. Although depressive disorders have been shown to be strongly associated with osteoporosis and increased fracture risk, evidence from post-traumatic stress disorder (PTSD) patients is less consistent. Therefore, the present study investigated the influence of chronic psychosocial stress on bone using a well-established murine model for PTSD. C57BL/6N mice (7 weeks old) were subjected to chronic subordinate colony housing (CSC) for 19 days, whereas control mice were singly housed. Anxiety-related behavior was assessed in the open-field/novel-object test, after which the mice were euthanized to assess endocrine and bone parameters. CSC mice exhibited increased anxiety-related behavior in the open-field/novel-object test, increased adrenal and decreased thymus weights, and unaffected plasma morning corticosterone. Microcomputed tomography and histomorphometrical analyses revealed significantly reduced tibia and femur lengths, increased growth-plate thickness and reduced mineral deposition at the growth plate, suggesting disturbed endochondral ossification during long-bone growth. This was associated with reduced Runx2 expression in hypertrophic chondrocytes in the growth plate. Trabecular thicknesses and bone mineral density were significantly increased in CSC compared to singly housed mice. Tyrosine hydroxylase expression was increased in bone marrow cells located at the growth plates of CSC mice, implying that local adrenergic signaling might be involved in the effects of CSC on the skeletal phenotype. In conclusion, chronic psychosocial stress negatively impacts endochondral ossification in the growth plate, affecting both longitudinal and appositional bone growth in adolescent mice. Summary: Chronic psychosocial stress is a known risk factor for many somatic disorders. The authors demonstrate that chronic psychosocial stress negatively affects endochondral ossification in the growth plate, as well as long-bone growth. This article has an associated First Person interview with the first author of the paper as part of the supplementary information.