Introduction: The influence of limb malrotation on long-leg radiographs (LLR) is frequently discussed in literature. This systematic review aimed to describe the influence of limb rotation on alignment measurements alone and in combination with knee flexion, and determine its clinical impact. Materials and methods: A literature search was conducted in June 2021 using the databases MEDLINE, Cochrane, Web of Science (Clarivate Analytics), and Embase. The search term ((radiograph OR X-ray) AND (position OR rotation) AND limb alignment) was used. Database query, record screening, and study inclusion and exclusion were performed by two reviewers independently. Experimental studies (using either specimens or synthetic bones) or clinical studies (prospective or retrospective using radiographs of patients) analyzing the influence of limb rotation on anatomic and mechanical limb alignment measurements were included. Characteristics and results of the included studies were summarized, simplified, and grouped for comparison to answer the research question. Studies were compared descriptively, and no meta-analysis was performed. Results: A total of 22 studies were included showing large heterogeneity, comprising studies with cadavers, patients, and synthetic bones. Most studies (7 out of 8) reported that external rotation (ER) causes less apparent valgus and leads to more varus and internal rotation (IR) causes more valgus and leads to less varus. However, there is no consensus on the extent of rotation influencing alignment measures. Studies reported about an average change of > 2° (n = 4) and < 2° (n = 4) hip-knee-ankle angle (HKA) between 15°IR and 15°ER. There is a consensus that the impact of rotation on mechanical alignment is higher if additional sagittal knee angulation, such as knee flexion, is present. All five studies analyzing the influence of rotation combined with knee flexion (5°–15°) showed an HKA change of > 2° between 15°IR and 15°ER. Conclusion: Malrotation is frequently present on LLR, possibly influencing the measured alignment especially in knees with extension deficit. Surgeons must consider this when measuring and treating deformities (high tibial osteotomy or total knee arthroplasties), and analyzing surgical outcomes. Especially in patients with osteoarthritis with knee extension deficits or postoperative swelling, the effect of malrotation is significantly greater.

Bone healing is a multifarious process involving mesenchymal stem cells, osteoprogenitor cells, macrophages, osteoblasts and -clasts, and chondrocytes to restore the osseous tissue. Particularly in long bones including the tibia, clavicle, humerus and femur, this process fails in 2–10% of all fractures, with devastating effects for the patient and the healthcare system. Underlying reasons for this failure are manifold, from lack of biomechanical stability to impaired biological host conditions and wound-immanent intricacies. In this review, we describe the cellular components involved in impaired bone healing and how they interfere with the delicately orchestrated processes of bone repair and formation. We subsequently outline and weigh the risk factors for the development of non-unions that have been established in the literature. Therapeutic prospects are illustrated and put into clinical perspective, before the applicability of biomarkers is finally discussed.

Co-culture models have become mandatory for obtaining better insights into bone homeostasis, which relies on the balance between osteoblasts and osteoclasts. Cigarette smoking (CS) has been proven to increase the risk of osteoporosis; however, there is currently no proven treatment for osteoporosis in smokers excluding cessation. Bisphosphonates (BPs) are classical anti-osteoclastic drugs that are commonly used in examining the suitability of bone co-culture systems in vitro as well as to verify the response to osteoporotic stimuli. In the present study, we tested the effects of BPs on cigarette smoke extract (CSE)-affected cells in the co-culture of osteoblasts and osteoclasts. Our results showed that BPs were able to reduce CSE-induced osteoporotic alterations in the co-culture of osteoblasts and osteoclasts such as decreased matrix remodeling, enhanced osteoclast activation, and an up-regulated receptor activator of nuclear factor (NF)-kB-ligand (RANKL)/osteoprotegerin (OPG) ratio. In summary, BPs may be an effective alternative therapy for reversing osteoporotic alterations in smokers, and the potential mechanism is through modulation of the RANKL/OPG ratio.

Smoking undoubtedly affects human health. Investigating 2318 representative patients at a level 1 trauma center identified delayed wound healing, tissue infections, and/or sepsis as main complications in smokers following trauma and orthopedic surgery. Therefore, smoking cessation is strongly advised to improve the clinical outcome in these patients, although smoking cessation often fails despite nicotine replacement therapy raising the need for specific interventions that may reduce the complication rate. However, the underlying mechanisms are still unknown. In diabetics, delayed wound healing and infections/sepsis are associated with increased neutrophilic PADI4 expression and formation of neutrophil extracellular traps (NETs). The aim was to investigate if similar mechanisms hold for smokers. Indeed, our results show higher PADI4 expression in active and heavy smokers than non-smokers, which is associated with an increased complication rate. However, in vitro stimulation of neutrophils with cigarette smoke extract (CSE) only moderately induced NET formation despite accumulation of reactive oxygen species (ROS). Physiological levels of nicotine and its main metabolite cotinine more effectively induced NET formation, although they did not actively induce the formation of ROS, but interfered with the activity of enzymes involved in anti-oxidative defense and NET formation. In summary, we propose increased formation of NETs as possible triggers for delayed wound healing, tissue infections, and/or sepsis in smokers after a major trauma and orthopedic surgery. Smoking cessation might reduce this effect. However, our data show that smoking cessation supported by nicotine replacement therapy should be carefully considered as nicotine and its metabolite cotinine effectively induced NET formation in vitro, even without active formation of ROS.

Many cells in the human body strongly react on decreased oxygen concentrations, generally defined as hypoxia. Therefore, inducing hypoxia in vitro is essential for research. Classically, hypoxia is induced using a hypoxia chamber, but alternative methods exist that do not require special equipment. Here, we compared three different methods to induce hypoxia without a hypoxia chamber: the chemical stabilization of HIF-1α by CoCl₂, the decrease in pericellular oxygen concentrations by increased media height, and the consumption of oxygen by an enzymatic system. Hypoxia induction was further analyzed within three different cell culture systems: 2D (adherent) osteoprogenitor cells, monocytic (suspension) cells, and in a 3D in vitro fracture hematoma model. The different methods were analyzed within the scope of fracture healing regarding inflammation and differentiation. We could show that all three induction methods were feasible for hypoxia induction within adherent cells. Increased media heights did not stimulate a hypoxic response within suspension cells and in the 3D system. Chemical stabilization of HIF-1α showed limitations when looking at the expression of cytokines in osteoprogenitors and monocytes. Enzymatic reduction of oxygen proofed to be most effective within all three systems inducing inflammation and differentiation.

Delayed fracture healing and fracture non-unions impose an enormous burden on individuals and society. Successful healing requires tight communication between immune cells and bone cells. Macrophages can be found in all healing phases. Due to their high plasticity and long life span, they represent good target cells for modulation. In the past, extremely low frequency pulsed electromagnet fields (ELF-PEMFs) have been shown to exert cell-specific effects depending on the field conditions. Thus, the aim was to identify the specific ELF-PEMFs able to modulate macrophage activity to indirectly promote mesenchymal stem/stromal cell (SCP-1 cells) function. After a blinded screening of 22 different ELF-PEMF, two fields (termed A and B) were further characterized as they diversely affected macrophage function. These two fields have similar fundamental frequencies (51.8 Hz and 52.3 Hz) but are emitted in different groups of pulses or rather send–pause intervals. Macrophages exposed to field A showed a pro-inflammatory function, represented by increased levels of phospho-Stat1 and CD86, the accumulation of ROS, and increased secretion of pro-inflammatory cytokines. In contrast, macrophages exposed to field B showed anti-inflammatory and pro-healing functions, represented by increased levels of Arginase I, increased secretion of anti-inflammatory cytokines, and growth factors are known to induce healing processes. The conditioned medium from macrophages exposed to both ELF-PEMFs favored the migration of SCP-1 cells, but the effect was stronger for field B. Furthermore, the conditioned medium from macrophages exposed to field B, but not to field A, stimulated the expression of extracellular matrix genes in SCP-1 cells, i.e., COL1A1, FN1, and BGN. In summary, our data show that specific ELF-PEMFs may affect immune cell function. Thus, knowing the specific ELF-PEMFs conditions and the underlying mechanisms bears great potential as an adjuvant treatment to modulate immune responses during pathologies, e.g., fracture healing.