The emerging role of tranexamic acid and its principal target, plasminogen, in skeletal health.

The worldwide burden of skeletal diseases such as osteoporosis, degenerative joint disease and impaired fracture healing is steadily increasing. Tranexamic acid (TXA), a plasminogen inhibitor and anti-fibrinolytic agent, is used to reduce bleeding with high effectiveness and safety in major surgical procedures. With its widespread clinical application, the effects of TXA beyond anti-fibrinolysis have been noticed and prompted renewed interest in its use. Some clinical trials have characterized the effects of TXA on reducing postoperative infection rates and regulating immune responses in patients undergoing surgery. Also, several animal studies suggest potential therapeutic effects of TXA on skeletal diseases such as osteoporosis and fracture healing. Although a direct effect of TXA on the differentiation and function of bone cells in vitro was shown, few mechanisms of action have been reported. Here, we summarize recent findings of the effects of TXA on skeletal diseases and discuss the underlying plasminogen-dependent and -independent mechanisms related to bone metabolism and the immune response. We furthermore discuss potential novel indications for TXA application as a treatment strategy for skeletal diseases.

Increased ß2-adrenergic signaling promotes fracture healing through callus neovascularization in mice.

Traumatic brain injury (TBI) leads to skeletal changes, including bone loss in the unfractured skeleton, and paradoxically accelerates healing of bone fractures; however, the mechanisms remain unclear. TBI is associated with a hyperadrenergic state characterized by increased norepinephrine release. Here, we identified the ß2-adrenergic receptor (ADRB2) as a mediator of skeletal changes in response to increased norepinephrine. In a murine model of femoral osteotomy combined with cortical impact brain injury, TBI was associated with ADRB2-dependent enhanced fracture healing compared with osteotomy alone. In the unfractured 12-week-old mouse skeleton, ADRB2 was required for TBI-induced decrease in bone formation and increased bone resorption. Adult 30-week-old mice had higher bone concentrations of norepinephrine, and ADRB2 expression was associated with decreased bone volume in the unfractured skeleton and better fracture healing in the injured skeleton. Norepinephrine stimulated expression of vascular endothelial growth factor A and calcitonin gene-related peptide-α (αCGRP) in periosteal cells through ADRB2, promoting formation of osteogenic type-H vessels in the fracture callus. Both ADRB2 and αCGRP were required for the beneficial effect of TBI on bone repair. Adult mice deficient in ADRB2 without TBI developed fracture nonunion despite high bone formation in uninjured bone. Blocking ADRB2 with propranolol impaired fracture healing in mice, whereas the ADRB2 agonist formoterol promoted fracture healing by regulating callus neovascularization. A retrospective cohort analysis of 72 patients with long bone fractures indicated improved callus formation in 36 patients treated with intravenous norepinephrine. These findings suggest that ADRB2 is a potential therapeutic target for promoting bone healing.

Tranexamic Acid Attenuates the Progression of Posttraumatic Osteoarthritis in Mice.

BACKGROUND: Posttraumatic osteoarthritis (OA) is a common disorder associated with a high socioeconomic burden, particularly in young, physically active, and working patients. Tranexamic acid (TXA) is commonly used in orthopaedic trauma surgery as an antifibrinolytic agent to control excessive bleeding. Previous studies have reported that TXA modulates inflammation and bone cell function, both of which are dysregulated during posttraumatic OA disease progression. PURPOSE: To evaluate the therapeutic effects of systemic and topical TXA treatment on the progression of posttraumatic OA in the knee of mice. STUDY DESIGN: Controlled laboratory study. METHODS: OA was induced via anterior cruciate ligament (ACL) transection on the right knee of female mice. Mice were treated with TXA or vehicle intraperitoneally daily or intra-articularly weekly for 4 weeks, starting on the day of surgery. Articular cartilage degeneration, synovitis, bone erosion, and osteophyte formation were scored histologically. Micro-computed tomography evaluation was conducted to measure the subchondral bone microstructure and osteophyte volume. Cartilage thickness and bone remodeling were assessed histomorphometrically. RESULTS: Both systemic and topical TXA treatment significantly reduced cartilage degeneration, synovitis, and bone erosion scores and increased the ratio of hyaline to calcified cartilage thickness in posttraumatic OA. Systemic TXA reversed ACL transection-induced subchondral bone loss and osteophyte formation, whereas topical treatment had no effect. Systemic TXA decreased the number and surface area of osteoclasts, whereas those of osteoblasts were not affected. No effect of topical TXA on osteoblast or osteoclast parameters was observed. CONCLUSION: Both systemic and topical TXA exerted protective effects on the progression of posttraumatic OA. Drug repurposing of TXA may, therefore, be useful for the prevention or treatment of posttraumatic OA, particularly after ACL surgery. CLINICAL RELEVANCE: TXA might be beneficial in patients with posttraumatic OA of the knee.

Transcript-dependent effects of the CALCA gene on the progression of post-traumatic osteoarthritis in mice.

Osteoarthritis represents a chronic degenerative joint disease with exceptional clinical relevance. Polymorphisms of the CALCA gene, giving rise to either a procalcitonin/calcitonin (PCT/CT) or a calcitonin gene-related peptide alpha (αCGRP) transcript by alternative splicing, were reported to be associated with the development of osteoarthritis. The objective of this study was to investigate the role of both PCT/CT and αCGRP transcripts in a mouse model of post-traumatic osteoarthritis (ptOA). WT, αCGRP-/- and CALCA-/- mice were subjected to anterior cruciate ligament transection (ACLT) to induce ptOA of the knee. Mice were sacrificed 4 and 8 weeks post-surgery, followed by micro-CT and histological evaluation. Here we show that the expression of both PCT/CT and αCGRP transcripts is induced in ptOA knees. CALCA-/- mice show increased cartilage degeneration and subchondral bone loss with elevated osteoclast numbers compared to αCGRP-/- and WT mice. Osteophyte formation is reduced to the same extent in CALCA-/- and αCGRP-/- mice compared to WT controls, while a reduced synovitis score is noticed exclusively in mice lacking CALCA. Our data show that expression of the PCT/CT transcript protects from the progression of ptOA, while αCGRP promotes osteophyte formation, suggesting that CALCA-encoded peptides may represent novel targets for the treatment of ptOA.

The selective norepinephrine reuptake inhibitor reboxetine promotes late-stage fracture healing in mice.

Impaired fracture healing is of high clinical relevance, as up to 15% of patients with long-bone fractures display non-unions. Fracture patients also include individuals treated with selective norepinephrine reuptake inhibitors (SNRI). As SNRI were previously shown to negatively affect bone homeostasis, it remained unclear whether patients with SNRI are at risk of impaired bone healing. Here, we show that daily treatment with the SNRI reboxetine reduces trabecular bone mass in the spine but increases cortical thickness and osteoblast numbers in the femoral midshaft. Most importantly, reboxetine does not impair bone regeneration in a standardized murine fracture model, and even improves callus bridging and biomechanical stability at late healing stages. In sum, reboxetine affects bone remodeling in a site-specific manner. Treatment does not interfere with the early and intermediate stages of bone regeneration and improves healing outcomes of the late-stage fracture callus in mice.

Increased beta2-adrenergic signaling is a targetable stimulus essential for bone healing by promoting callus neovascularization.

Traumatic brain injury (TBI) is associated with a hyperadrenergic state and paradoxically causes systemic bone loss while accelerating fracture healing. Here, we identify the beta2-adrenergic receptor (Adrb2) as a central mediator of these skeletal manifestations. While the negative effects of TBI on the unfractured skeleton can be explained by the established impact of Adrb2 signaling on bone formation, Adrb2 promotes neovascularization of the fracture callus under conditions of high sympathetic tone, including TBI and advanced age. Mechanistically, norepinephrine stimulates the expression of Vegfa and Cgrp primarily in periosteal cells via Adrb2, both of which synergistically promote the formation of osteogenic type-H vessels in the fracture callus. Accordingly, the beneficial effect of TBI on bone repair is abolished in mice lacking Adrb2 or Cgrp, and aged Adrb2-deficient mice without TBI develop fracture nonunions despite high bone formation in uninjured bone. Pharmacologically, the Adrb2 antagonist propranolol impairs, and the agonist formoterol promotes fracture healing in aged mice by regulating callus neovascularization. Clinically, intravenous beta-adrenergic sympathomimetics are associated with improved callus formation in trauma patients with long bone fractures. Thus, Adrb2 is a novel target for promoting bone healing, and widely used beta-blockers may cause fracture nonunion under conditions of increased sympathetic tone.

The role of sphingosine-1-phosphate in bone remodeling and osteoporosis.

Osteoporosis is a systemic bone disease that affects more than 200 million people worldwide and is caused by the disruption of the equilibrium between osteoclastic bone resorption and osteoblastic bone formation. Sphingosine-1-phosphate (S1P) is a natural, bioactive sphingolipid that has been shown to play a major role in cardiovascular and immunological pathologies by regulating biological and cellular processes, including migration, differentiation, proliferation and survival. Recent studies also suggest a central role for S1P in bone diseases, including osteoporosis; however, the effects of S1P, particularly in bone metabolism, remain to be further elucidated. In this review, we summarize the available literature on the role of S1P in bone metabolism with a focus on osteoporosis. On the cellular level, S1P acts as an osteoclast-osteoblast coupling factor to promote osteoblast proliferation and bone formation. Moreover, the recruitment of osteoclast precursors to resorption sites is regulated by the interplay of S1P gradients and S1P receptor expression. From a clinical perspective, increasing evidence suggests that systemically elevated S1P blood levels may serve as an independent risk factor for osteoporosis-related fractures. Taken together, S1P signaling is a potential therapeutic target and may serve as a novel biomarker in patients with systemic bone disease.

Systemic calcitonin gene-related peptide receptor antagonism decreases survival in a porcine model of polymicrobial sepsis: blinded randomised controlled trial.

BACKGROUND: Calcitonin gene-related peptide (CGRP) and procalcitonin, which are overexpressed in sepsis, exert distinct immunomodulatory effects mediated through the CGRP receptor. The CGRP receptor antagonist olcegepant improves survival in murine sepsis. This study evaluated whether CGRP receptor antagonism is similarly beneficial in a porcine model of polymicrobial sepsis. METHODS: We conducted a prospective randomised, controlled, investigator-blinded trial in adult pigs of either sex, that were anaesthetised and ventilated before sepsis was induced by polymicrobial (autologous) faecal peritonitis. After the onset of early septic shock (systolic blood pressure <90 mm Hg or >10% decline from baseline MAP), pigs were resuscitated (i.v. fluid/antibiotics/vasopressors) and randomised to receive either i.v. olcegepant (n=8) or vehicle control (n=8). The primary outcome was time to death, euthanasia required up to 72 h after surgery (according to predefined severe cardiorespiratory failure), or both. Secondary outcomes included haemodynamic changes, and systemic as well as organ inflammation (mRNA expression). RESULTS: Septic shock developed 8.7 h (inter-quartile range, 5.8-11.1 h) after the onset of faecal peritonitis. Olcegepant worsened survival, with 6/8 pigs randomised to the control group surviving 72.0 h (50.9-72.0 h), compared with 3/8 pigs receiving olcegepant surviving 51.3 h (12.5-72.0 h; P=0.01). At 48 h, lower MAP and higher cardiac output occurred in pigs receiving olcegepant. Cardiac, hepatic, and renal injury was not different between pigs randomised to receive olcegepant or vehicle. Olcegepant reduced mRNA expression of several inflammation-related cytokines and CD68+ macrophages in liver but not in lung tissue. CONCLUSIONS: CGRP receptor antagonism with olcegepant was not beneficial in this porcine model of polymicrobial sepsis, which closely mimics human sepsis.

Procalcitonin is expressed in osteoblasts and limits bone resorption through inhibition of macrophage migration during intermittent PTH treatment.

Intermittent injections of parathyroid hormone (iPTH) are applied clinically to stimulate bone formation by osteoblasts, although continuous elevation of parathyroid hormone (PTH) primarily results in increased bone resorption. Here, we identified Calca, encoding the sepsis biomarker procalcitonin (ProCT), as a novel target gene of PTH in murine osteoblasts that inhibits osteoclast formation. During iPTH treatment, mice lacking ProCT develop increased bone resorption with excessive osteoclast formation in both the long bones and axial skeleton. Mechanistically, ProCT inhibits the expression of key mediators involved in the recruitment of macrophages, representing osteoclast precursors. Accordingly, ProCT arrests macrophage migration and causes inhibition of early but not late osteoclastogenesis. In conclusion, our results reveal a potential role of osteoblast-derived ProCT in the bone microenvironment that is required to limit bone resorption during iPTH.

An optimized protocol for a standardized, femoral osteotomy model to study fracture healing in mice.

Fracture healing represents a dynamic and complex process which depends on the balanced activities of a broad array of different cell types and signaling pathways. Here, we describe a femoral osteotomy protocol for mice, using an external fixator to stabilize the fractured bone. Depending on experimental requirements, the size of the osteotomy gap can be adjusted. Taken together, this protocol describes a highly standardized and reproducible murine model for morphologic and biomolecular assessment of the fracture healing process. For complete details on the use and execution of this protocol, please refer to Appelt et al. (2020).

Glucose Metabolism in Osteoblasts in Healthy and Pathophysiological Conditions.

Bone tissue in vertebrates is essential to performing movements, to protecting internal organs and to regulating calcium homeostasis. Moreover, bone has also been suggested to contribute to whole-body physiology as an endocrine organ, affecting male fertility; brain development and cognition; and glucose metabolism. A main determinant of bone quality is the constant remodeling carried out by osteoblasts and osteoclasts, a process consuming vast amounts of energy. In turn, clinical conditions associated with impaired glucose metabolism, including type I and type II diabetes and anorexia nervosa, are associated with impaired bone turnover. As osteoblasts are required for collagen synthesis and matrix mineralization, they represent one of the most important targets for pharmacological augmentation of bone mass. To fulfill their function, osteoblasts primarily utilize glucose through aerobic glycolysis, a process which is regulated by various molecular switches and generates adenosine triphosphate rapidly. In this regard, researchers have been investigating the complex processes of energy utilization in osteoblasts in recent years, not only to improve bone turnover in metabolic disease, but also to identify novel treatment options for primary bone diseases. This review focuses on the metabolism of glucose in osteoblasts in physiological and pathophysiological conditions.