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Abstract Background Biomaterials used in fracture healing hold a significant place in orthopedics. This study aimed to develop biomaterials coated with hydroxyapatite (HA), boric acid (BA), and magnesium (Mg) and investigate their effects on fracture healing. Methods Sixty female Wistar Albino rats were included in the study. The subjects were randomized into five groups. Cytotoxicity tests were performed on HA, BA, and Mg, and cell viability rates were calculated. Coatings were applied to Kirschner (K) wires at determined ratios. Group I was the control group with a steel K wire, Group II used HA-coated K wires, Group III used HA + BA-coated K wires, Group IV used HA + BA + Mg-coated K wires, and Group V used HA + Mg-coated K wires. A fracture was induced in the right femur of the subjects, followed by fixation with intramedullary K wires. The subjects were randomly divided into equal numbers and sacrificed at 6 and 12 weeks. Radiological and histopathological evaluations were performed. Results In direct cytotoxicity tests, the highest viability rate was observed in Group IV, while in indirect cytotoxicity tests, it was highest in Group II. In radiological evaluation at the 6th week, the highest scores were in Groups IV and V, while the lowest was in Group III. At the 12th week, the highest scores were in Groups II and V, while the lowest was in Group I. No significant differences were found between the groups (p = 0.837, p = 0.0479). In histopathological evaluation, a significant difference was observed between the groups (p

Abstract Background Low-intensity pulsed ultrasound (LIPUS) is a non-invasive therapy that accelerates fracture healing. As a new treatment method for fracture, we recently reported that the transcutaneous application of CO2 accelerated fracture healing in association with promoting angiogenesis, blood flow, and endochondral ossification. We hypothesized that transcutaneous CO2 application, combined with LIPUS, would promote bone fracture healing more than the single treatment with either of them. Methods Femoral shaft fractures were produced in 12-week-old rats. Animals were randomly divided into four groups: the combination of CO2 and LIPUS, CO2, LIPUS, and control groups. As the transcutaneous CO2 application, the limb was sealed in a CO2-filled bag after applying hydrogel that promotes CO2 absorption. Transcutaneous CO2 application and LIPUS irradiation were performed for 20 min/day, 5 days/week. At weeks 1, 2, 3, and 4 after the fractures, we assessed the fracture healing process using radiography, histology, immunohistochemistry, real-time PCR, and biomechanical assessment. Results The fracture healing score using radiographs in the combination group was significantly higher than that in the control group at all time points and those in both the LIPUS and CO2 groups at weeks 1, 2, and 4. The degree of bone fracture healing in the histological assessment was significantly higher in the combination group than that in the control group at weeks 2, 3, and 4. In the immunohistochemical assessment, the vascular densities of CD31- and endomucin-positive microvessels in the combination group were significantly higher than those in the control and LIPUS groups at week 2. In the gene expression assessment, significant upregulation of runt-related transcription factor 2 (Runx2) and vascular endothelial growth factor (VEGF) was detected in the combination group compared to the LIPUS and CO2 monotherapy groups. In the biomechanical assessment, the ultimate stress was significantly higher in the combination group than in the LIPUS and CO2 groups. Conclusion The combination therapy of transcutaneous CO2 application and LIPUS had a superior effect in promoting fracture healing through the promotion of angiogenesis and osteoblast differentiation compared to monotherapy.

Aims: This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels. Methods: A closed tibial fracture model was established using 12 male Japanese white rabbits, and divided into elastic and stiff fixation groups based on different fixation methods. Two weeks after the operation, a radiograph and pathological examination of callus tissue were used to evaluate fracture healing. Then, the differentially expressed proteins (DEPs) were examined in the callus using proteomics. Finally, in vitro cell experiments were conducted to investigate hub proteins involved in this process. Results: Mean callus volume was larger in the elastic fixation group (1,755 mm3 (standard error of the mean (SEM) 297)) than in the stiff fixation group (258 mm3 (SEM 65)). Pathological observation found that the expression levels of osterix (OSX), collagen, type I, alpha 1 (COL1α1), and alkaline phosphatase (ALP) in the callus of the elastic fixation group were higher than those of the stiff fixation group. The protein sequence of the callus revealed 199 DEPs, 124 of which were highly expressed in the elastic fixation group. In the in vitro study, it was observed that a stress of 200 g led to upregulation of thrombospondin 1 (THBS1) and osteoglycin (OGN) expression in bone marrow mesenchymal stem cells (BMSCs). Additionally, these genes were found to be upregulated during the osteogenic differentiation process of the BMSCs. Conclusion: Elastic fixation can promote fracture healing and osteoblast differentiation in callus, and the ability of elastic fixation to promote osteogenic differentiation of BMSCs may be achieved by upregulating genes such as THBS1 and OGN. Cite this article: Bone Joint Res 2024;13(10):559–572.

Abstract Fracture healing is usually monitored by clinical impressions and radiographs. Objective and easy methods for assessing fracture healing without radiation would be beneficial. The aim of this study was to analyse whether weight and plantar pressure while standing can be used to monitor healing of tibial or malleolar fractures and whether these parameters can discriminate between patients with and without union. Thirteen patients were longitudinally assessed during each postoperative clinical visit, of whom two developed a nonunion. Eleven matched healthy controls were assessed once. Additionally, five patients already experiencing nonunion were assessed once at the time of their nonunion diagnosis. All participants performed a standing task for ten seconds with pressure-sensing insoles. Greatest improvements were detected throughout the first three months in patients with union. However, six months after surgery, more than half of the parameters were still significantly different from those of the controls. The weight and pressure distributions did not differ between patients with or without union six months after surgery. A standing task can be used to monitor improvements in weight and pressure distribution throughout the healing process of tibial or malleolar fractures, but lacks potential to discriminate between patients with or without fracture union.

Abstract Background Prompt identification of fracture healing is crucial for medical decision-making and clinical research. This study aims to assess the effectiveness of the Radiographic Union Score for Hip (RUSH) in subtrochanteric fractures and determine the optimal scoring threshold for fracture healing. Methods We obtained 94 sets of X-ray films from the postoperative follow-up of 35 patients who underwent intramedullary nail fixation for subtrochanteric fractures. Six orthopedic trauma surgeons evaluated the imaging data and determined fracture healing based on subjective judgment. They then scored the X-ray images using the RUSH form. After four weeks, the same observers re-evaluated 47 randomly selected sets of radiographs. Subsequently, the observers and study designer conducted a consensus meeting where they agreed on whether the fractures had healed. We used Fleiss kappa to evaluate agreement among observers based on subjective impressions, and the intraclass correlation coefficient assessed RUSH score consistency. Regression analysis examined the relationship between scores and fracture healing consensus using a receiver operator characteristic (ROC) curve. Results Based on overall impressions, the agreement level among orthopedic trauma surgeons for determining the healing status of subtrochanteric fractures was moderate (Kappa = 0.564, 95% CI: 0.511–0.616). However, utilizing the RUSH scoring system can improve the consistency to a substantial level of agreement (ICC = 0.748, 95% CI: 0.663–0.817). Regarding intraobserver reliability, there were significant differences among orthopedic trauma surgeons in judging fracture healing based on overall impressions. However, using the RUSH scoring form, the attending group achieved better intraobserver consistency than the resident group. Regression analysis and ROC curve analysis revealed a strong correlation between the RUSH total score, cortical bone score, cancellous bone score, and consensus on fracture healing. The AUC values are 0.769 (95% CI: 0.670–0.868), 0.779 (95% CI: 0.681–0.876), and 0.771 (95% CI: 0.674–0.867), respectively. Conclusions Our study highlights the effectiveness of the RUSH scoring system in enhancing interobserver agreement and intraobserver consistency when evaluating subtrochanteric fracture healing in orthopedic trauma surgeons. We propose setting 21 points as the threshold for the simplified RUSH scoring system to determine fracture healing. This system includes only eight items related to cortical bone, with a total score of 24 points.

Abstract To assess the efficacy of a novel 3D biomimetic hydrogel scaffold with immunomodulatory properties in promoting fracture healing. Immunomodulatory scaffolds were used in cell experiments, osteotomy mice treatment, and single-cell transcriptomic sequencing. In vitro, fluorescence tracing examined macrophage mitochondrial transfer and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Scaffold efficacy was assessed through alkaline phosphatase (ALP), Alizarin Red S (ARS) staining, and in vivo experiments. The scaffold demonstrated excellent biocompatibility and antioxidant-immune regulation. Single-cell sequencing revealed a shift in macrophage distribution towards the M2 phenotype. In vitro experiments showed that macrophage mitochondria promoted BMSCs’ osteogenic differentiation. In vivo experiments confirmed accelerated fracture healing. The GAD/Ag-pIO scaffold enhances osteogenic differentiation and fracture healing through immunomodulation and promotion of macrophage mitochondrial transfer.

Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing. In this study, we aimed to develop an osteoanabolic therapy which activates the Wnt/β-catenin pathway, a molecular driver of endochondral ossification. We hypothesize that using an mRNA-based therapeutic encoding β-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes. To optimize a delivery platform built on recent advancements in liposomal technologies, two FDA-approved ionizable phospholipids, DLin-MC3-DMA (MC3) and SM-102, were used to fabricate unique ionizable lipid nanoparticle (LNP) formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model. Using firefly luciferase mRNA as a reporter gene to track and quantify transfection, SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection, minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs. The generated β-cateninGOF mRNA encapsulated in SM-102 LNPs (SM-102-β-cateninGOF mRNA) showed bioactivity in vitro through upregulation of downstream canonical Wnt genes, axin2 and runx2. When testing SM-102-β-cateninGOF mRNA therapeutic in a murine tibia fracture model, histomorphometric analysis showed increased bone and decreased cartilage composition with the 45 μg concentration at 2 weeks post-fracture. μCT testing confirmed that SM-102-β-cateninGOF mRNA promoted bone formation in vivo, revealing significantly more bone volume over total volume in the 45 μg group. Thus, we generated a novel mRNA-based therapeutic encoding a β-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery.

Objective To investigate the effect of recombinant human lactoferrin (rhLF) on fracture healing in rat models with femur fracture and the proliferation of rat primary osteoblasts. Methods Male rats were randomly divided into 3 groups: simple fracture group (SF group), collagen film-treated group (CF group) and rhLF-compound collagen lamination film-treated group (CLF group). The fracture healing was observed by X-ray image and histological microscopy with HE and Masson staining. The newborn SD rats with osteoblasts were treated with different concentrations of rhLF, and osteoblast proliferation was detected by MTT method. Results The bone scabs in the CLF group were significantly larger than those in the SF and CF groups at 1, 2, and 3 weeks postoperatively (P＜0.05). There was no difference in the comparison between the groups at 4th week. The proliferation of cartilaginous and sclerotic bone scabs in the CLF group was more pronounced and showed the appearance of mature trabeculae and lamellar bone with a high degree of maturation of the bone scabs. The groups with 100 mg/L and 500 mg/L rhLF showed a significant decrease in A value for rat primary osteoblasts cultured in vitro as compared with the control group(P＜0.05). The A value increased significantly at the first day (P＜0.05). The concentration of rhLF increased at the fifth and tenth days. Conclusions rhLF has an accelerating effect on fracture healing which is manifested by fast scab formation, large volume and high maturity. The rhLF can promote the proliferation of osteoblasts cultured in vitro, and the effect is mainly manifested in the rapid proliferation stage of osteoblasts.

Introduction: Non-union of long bones is a common challenge in the treatment of fractures. Bone grafting is commonly used to treat atrophic non-union, but mechanical displacement of the graft may occur, resulting in delay or failure of treatment. Fibrin glue has demonstrated positive results in management of bone defects in neurosurgery and oromaxillary facial surgery, however, there has yet to be any study on its use in long bone fractures. Materials and methods: We conducted a prospective randomised controlled trial at a single tertiary centre involving adult patients with long bone fractures that had undergone non-union and requiring bone grafting only. Autologous iliac crest bone graft was applied to the debrided non-union site, with additional fibrin glue applied for the intervention arm. Patients were followed-up with serial radiographs until clinical and radiographical union. Results: Ten patients (3 male, 7 female), of mean age 41.7 (19 – 63) were recruited over five years, with one drop out. Eight out of nine fractures united after treatment. One patient underwent hypertrophic non-union requiring re-fixation and bone grafting. There was no difference in the time to union for patients in the fibrin glue group (19.5 weeks) versus the control group (18.75 weeks) (p=0.86). There were no complications sustained from usage of fibrin glue. Conclusions: Fibrin glue appears to be a safe adjunct for treatment of non-union of long bone fractures across varying fracture sites by holding the bone graft in place despite not demonstrating a faster time to union.

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.

Traumatic fractures and stress fractures are common orthopedic diseases, and there is great potential in researching bone turnover, repair, and promotion of fracture healing. Basic medical experiments often use animal models of long bone fractures in limbs to study the mechanisms of various interventions on fracture healing. Fracture healing is a complex process influenced by multiple factors and involves multiple molecules and pathways. Therefore, to explore the mechanisms more deeply, accelerate the translation of results, and improve the clinical efficacy, it is particularly important to choose the appropriate animal fracture modeling methods in experimental research. Based on this, this paper conducts a literature review of animal species and modeling methods commonly used for long bone fracture models in experimental research. It summarizes five methods: bone defect method, physical impact method, mechanical bending method, open osteotomy method, and drilling method. A side-by-side comparison of their advantages, disadvantages, and scope of application is made, aiming to provide suitable fracture models for studying the mechanisms of fracture healing interventions.

Fracture healing is a complex event that involves the coordination of various different processes, including intramembranous and endochondral bone formation. When facing fracture nonunion or delayed union, few organizational engineering structures can achieve the desired results. The main reason for this is that they cannot recapitulate the cellular morphology, biology, and mechanical functions of natural tissues. Ten years ago, the term Development Engineering was coined to refer to the use of developmental processes as a blueprint for designing and developing engineered live implants. Different sources of cells have been used as seed cells in developmental engineering. Among them, hypertrophic chondrocytes have attracted worldwide attention. Hypertrophic chondrocytes are the terminal state of growth plate chondrocytes, leading to degenerative maturation. Hypertrophic chondrocytes mediate crosstalk by regulating cell-matrix degradation, vascularization, osteoclast recruitment, and osteoblast differentiation. Furthermore, hypertrophic chondrocytes can transdifferentiate into osteoprogenitors and mature osteoblasts, and directly promote woven bone formation. In summary, elucidating the role of hypertrophic chondrocytes will contribute to understand of the physiological mechanism of fracture healing, research and development of novel therapeutic modes of developmental engineering, and further promotion of fracture healing.

Abstract Background Long non-coding RNAs (LncRNAs) are recognized as a pivotal element in the processes of fracture healing and the osteogenic differentiation of stem cells. This study investigated the molecular mechanism and regulatory significance of lncRNA MAGI2-AS3 (MAGI2-AS3) in fracture healing. Methods Serum levels of MAGI2-AS3 in patients with normal and delayed fracture healing were verified by RT-qPCR assays. The predictive efficacy of MAGI2-AS3 for delayed fracture healing was analyzed by ROC curve. Osteogenic markers were quantified by RT-qPCR assays. MC3T3-E1 cell viability was detected using CCK-8 assay, and flow cytometry was utilized to measure cell apoptosis. The dual-luciferase reporter gene assay was used to determine the targeted binding between MAGI2-AS3 and miR-223-3p. Results Serum MAGI2-AS3 expression was decreased in patients with delayed fracture healing compared with patients with normal healing. Elevated MAGI2-AS3 resulted in an upregulation of the proliferative capacity of MC3T3-E1 cells and a decrease in mortality, along with increased levels of both osteogenic markers. However, after transfection silencing MAGI2-AS3, the trend was reversed. Additionally, miR-223-3p was the downstream target of MAGI2-AS3 and was controlled by MAGI2-AS3. miR-223-3p mimic reversed the promoting effects of MAGI2-AS3 overexpression on osteogenic marker levels and cell growth, and induced cell apoptosis. Conclusion The upregulation of MAGI2-AS3 may expedite the healing of fracture patients by targeting miR-223-3p, offering a novel biomarker for diagnosing patients with delayed healing.

Abstract Background Fragility fracture is common in the elderly. Osteoblast differentiation is essential for bone healing and regeneration. Expression pattern of long non-coding RNA MIAT during fracture healing was examined, and its role in osteoblast differentiation was investigated. Methods 90 women with simple osteoporosis and 90 women with fragility fractures were included. Another 90 age-matched women were set as the control group. mRNA levels were tested using RT-qPCR. Cell viability was detected via CCK-8, and osteoblastic biomarkers, including ALP, OCN, Collagen I, and RUNX2 were tested via ELISA. The downstream miRNAs and genes targeted by MIAT were predicted by bioinformatics analysis, whose functions and pathways were annotated via GO and KEGG analysis. Results Serum MIAT was upregulated in osteoporosis women with high accuracy of diagnostic efficacy. Serum MIAT was even elevated in the fragility fracture group, but decreased in a time manner after operation. MIAT knockdown promoted osteogenic proliferation and differentiation of MC3T3-E1, but the influences were reversed by miR-181a-5p inhibitor. A total of 137 overlapping target genes of miR-181a-5p were predicted based on the miRDB, TargetScan and microT datasets, which were mainly enriched for terms related to signaling pathways regulating pluripotency of stem cells, cellular senescence, and osteoclast differentiation. Conclusions LncRNA MIAT serves as a promising biomarker for osteoporosis, and promotes osteogenic differentiation via targeting miR-181a-5p.

Objective: To evaluate the role of autologous adipose-derived stem cells (ADSCs) with exogenous neuropeptide Y (NPY) on osteoporotic fracture healing. Methods: We established a fracture healing model in ovariectomized rats of osteoporosis. Autologous ADSCs, isolated from rats' subcutaneous adipose tissue, with exogenous NPY were transplanted into the fracture site four times a week. The fracture healing was observiked with X-ray diagnostic techniques, and the rats’ tibias were taken out for section-staining and micro-CT scanning 3 and 6 weeks after fracture. Next, we explored the underlying mechanism by which the transplantation contributed to fracture healing. Results: Co-culture and co-transplantation of ADSCs with NPY could stimulate the osteoporotic fracture healing. NPY may promote the osteogenic differentiation of ADSCs through Y1 receptor. Conclusion: This result provides a platform for the development of novel therapies for bone regeneration with endogenous ADSCs.

Objective: To examine and talk about the mechanism of the Huoxue Jiegu compound capsule's effects on osteoblasts and the PI3K/Akt/mTOR signal pathway in rabbits suffering from tibial fractures. Method: In vitro, CCK8 was used to assess the survival rates. Alizarinred staining was used to evaluate mineralized nodules. ALP staining was used to observe the osteoblasts. qRT-PCR was used to determine the mRNA expression of the bone formation-related factors BMP-2, bFGF, and TGF-β. In vivo, three groups of nine male rabbits each were randomly assigned to three groups: the Model group, the Huoxue Jiegu compound capsule group (HXJGC group), and the inhibitor group (HXJGC+3-MA), four weeks following the intervention. HE staining was employed to examine the rabbits' bone histology. immunohistochemistry was employed to examine the relative expression of the proteins VEGF and LC3-II. Western Blot was utilized to examine the relative expression of the proteins Beclin-1, LC3-II/Ⅰ, p62, p-PI3K, p-AKT, and p-mTOR. Results: Compared to the control group, the medium- and high-dose groups exhibited considerably higher survival rates (P

Abstract Mesenchymal stem cells are core to bone homeostasis and repair. They both provide the progenitor cells from which bone cells are formed and regulate the local cytokine environment to create a pro-osteogenic environment. Dysregulation of these cells is often seen in orthopaedic pathology and can be manipulated by the physician treating the patient. This narrative review aims to describe the common applications of cell therapies to bone healing whilst also suggesting the future direction of these techniques.

Abstract Background External fixation is widely used in the treatment of traumatic fractures; however, orthopedic surgeons encounter challenges in deciding the optimal time for fixator removal. The axial load-share ratio (LS) of the fixator is a quantitative index to evaluate the stiffness of callus healing. This paper introduces an innovative method for measuring the LS and assesses the method’s feasibility and efficacy. Based on a novel hexapod LS-measurement system, the proposed method is to improve the convenience and precision of measuring LS in vivo, hence facilitating the safe removal of external fixators. Methods A novel hexapod system is introduced, including its composition, theoretical model, and method for LS measurement. We conducted a retrospective study on 82 patients with tibial fractures treated by the Taylor Spatial Frame in our hospital from September 2018 to June 2020, of which 35 took LS measurements with our novel method (Group I), and 47 were with the traditional method (Group II). The external fixator was removed when the measurement outcome (LS 0.05), frame-wearing duration (mean 25.5 weeks vs. 25.8 weeks, P > 0.05), or LS measurement frequency (mean 1.1 times vs. 1.2 times, P > 0.05). The measurement system installation time in Group I was significantly shorter compared to Group II (mean 14.8 min vs. 81.3 min, P 0.05). Conclusion The novel hexapod LS-measurement system and involved method demonstrated enhanced convenience and precision in measuring the LS of the external fixator in vivo. The LS measurement indicates the callus stiffness of fracture healing, and is applicable to evaluate the safety of removing the fixator. Consequently, it is highly recommended for widespread adoption in clinical practice.

Introduction/purpose: SMART orthopedic systems use fixators with remote monitoring, processing, and communication capabilities to leverage healing progression data for personalized, real-time monitoring of a healing process. The fixators incorporate small and compact piezoelectric sensors that generate electrical signals upon the application of force to the piezoelectric diaphragm. This enables doctors to remotely guide fixation devices using indirectly and remotely controlled stepper motors known for their precision and accuracy. Reliability of stepper motors makes them a viable alternative for the mechanical tools traditionally used by doctors for fixator extension. Methods: This study focuses on the evaluation of sensor-based technology in orthopedic applications. The paper presents a theoretical framework for the application of SMART devices in the bone fracture healing process. It delves into the structure and functionality of piezoelectric transducers, offering a comprehensive insight into this technology and various engineering aspects of SMART systems. Results: The implementation of SMART systems has significantly enhanced doctor-patient communication. This improvement is facilitated through a dual-phase process involving gathering, processing, and transmitting the data wirelessly from the patient's (sensor) interface to the doctor who uses specialized software for data analysis and wireless transmission to the stepper motor actuator. Subsequently, the data is forwarded to the decoder at the motor site, where a motor controller generates the control signal for the stepper motor driver. Conclusion: SMART implants can provide doctors with quantitative data that can be used in directing a rehabilitation plan. The sensor-based technology offers insights into the stress induced by the callus formation enabling bidirectional communication between the doctor and the patient. The stepper motor is a tool that aids in personalized treatment from the distance.

Magnetic fields are safe and used in noninvasive physical therapies. Numerous studies have confirmed that magnetic fields have good osteogenic effects and certain value for clinical application in accelerating orthodontic tooth movement, promoting bone-implant integration, promoting fracture healing and improving the effects of distraction osteogenesis. Magnetic fields are expected to become applied as effective auxiliary methods for treating oral diseases. To support the clinical application of magnetic fields, this article reviews the applications of magnetic fields in the oral cavity, the biological effects on bone cells and the molecular mechanisms through which magnetic fields regulate bone metabolism. The biological effects of magnetic fields on bone cells include promoting osteogenesis by osteoblasts and mesenchymal stem cells and inhibiting bone resorption by osteoclasts. At the molecular level, bone cells sense and respond to magnetic stimulation, and through various mechanisms, such as displacement currents, Lorentz forces, and free radical pair effects, stimuli are transformed into biologically recognizable electrical signals that activate complex downstream signaling pathways, such as the P2 purinergic receptor signaling pathway, adenosine receptor signaling pathway, transforming growth factor-β receptor signaling pathway, mammalian target of rapamycin (mTOR) pathway, and Notch pathway. In addition, magnetic parameters, which are the factors affecting the osteogenic effects of magnetic fields, are discussed. However, the mechanisms of the osteogenic effects of magnetic fields are unclear, and further studies of these mechanisms could provide effective strategies for bone regeneration and periodontal tissue regeneration. In addition, considering the target of magnetic field therapies, combination with other drugs could lead to new strategies for the treatment of oral diseases.

Abstract Background Collarbone fracture is a common injury, particularly among athletes involved in contact sports and participating in endurance activities. Conventional treatment requires surgery and postoperative immobilization, resulting in an average return-to-sport timeframe of approximately 13 weeks. This case challenges the established treatment protocols, aiming to expedite recovery and enable a quicker resumption of high-intensity athletic activities. Case presentation A 24-year-old Caucasian athlete completed a Half-Ironman Triathlon (70.3) merely three weeks post-collarbone fracture. Utilizing Extracorporeal Magneto-Transduction Therapy (EMTT) alongside surgical intervention, the patient achieved accelerated healing and remarkable performance outcomes without encountering any adverse effects. Conclusions The integration of EMTT into the treatment paradigm for bone fractures alters the traditional understanding of recovery timelines and rehabilitation strategies. This case highlights the potential benefits of electromagnetic wave therapy in expediting the healing process and enabling athletes to resume high-level sports activities at an earlier stage.

Fracture healing is a complex staged repair process in which the mechanical environment plays a key role. Bone tissue is very sensitive to mechanical stress stimuli, and the literature suggests that appropriate stress can promote fracture healing by altering cellular function. However, fracture healing is a coupled process involving multiple cell types that balance and limit each other to ensure proper fracture healing. The main cells that function during different stages of fracture healing are different, and the types and molecular mechanisms of stress required are also different. Most previous studies have used a single mechanical stimulus on individual mechanosensitive cells, and there is no relatively uniform standard for the size and frequency of the mechanical stress. Analyzing the mechanisms underlying the effects of mechanical stimulation on the metabolic regulation of signaling pathways in cells such as in bone marrow mesenchymal stem cells (BMSCs), osteoblasts, chondrocytes, and osteoclasts is currently a challenging research hotspot. Grasping how stress affects the function of different cells at the molecular biology level can contribute to the refined management of fracture healing. Therefore, in this review, we summarize the relevant literature and describe the effects of mechanical stress on cells associated with fracture healing, and their possible signaling pathways, for the treatment of fractures and the further development of regenerative medicine.

Background: Recently, the osteogenic potential of Adiponectin-labeled adipogenic lineage progenitors (Adipoq-lineage progenitors) in bone marrow has been observed to support bone maintenance and repair. However, little is known about the function of Schnurri-3 (SHN3, also known as HIVEP3) in other mesenchymal lineage cells, apart from its negative regulation of bone formation on osteoblasts. Method: In this study, we used single-cell RNA sequencing (scRNA-seq) profiling to demonstrate that Adipoq-lineage progenitors express higher levels of Shn3 compared to other mesenchymal cell populations in mice and humans. To investigate the role of SHN3 in Adipoq-lineage progenitors, we generated a murine model specifically harboring a Shn3-deficient allele in Adipoq-expressing cells. Information of mice body weight was collected weekly to generate body weight curve. Bone phenotype was analyzed using micro-CT and histomorphometric studies. To eliminate the role of peripheral adipose tissue on bone, we collected adipose wet weight, performed intraperitoneal glucose tolerance tests and intraperitoneal insulin tolerance tests, and conducted a fat-transplantation study. Osteoblast and osteoclast functions were assessed through toluidine blue staining and TRAP staining, respectively. We further investigated the effect of Shn3 depletion on the differentiation of Adipoq-lineage progenitors through immunostaining and in vitro differentiation assays. Finally, we evaluated whether Shn3 deficiency in Adipoq-lineage progenitors affects the fracture healing process by generating bi-cortical femoral fracture models. Results: Depletion of Shn3 in Adipoq-lineage progenitors resulted in a significant increase in trabecular bone mass and bone formation in vivo, without disrupting whole-body energy metabolism and skeletal development. Consistent with these findings, both cell-lineage tracing and functional assays revealed that Shn3 ablation effectively shifted the cell fate of Adipoq-lineage progenitors towards an osteogenic phenotype in the bone marrow. Furthermore, in vivo studies demonstrated that the lack of Shn3 in Adipoq-lineage progenitors also enhanced bone fracture healing under pathological conditions. Conclusion: Overall, our findings provide a novel strategy for targeting the osteoanabolic potential of bone marrow Adipoq-lineage progenitors as a potential treatment for bone loss-related disorders. Translational potential of this article: We have identified a novel gene target that directs the cell fate of a previously identified non-osteogenic cell population under physiological conditions. This study not only expands the therapeutic value of Shn3 ablation in treating osteoporotic or traumatic bone diseases but also provides new insights into the contribution of bone marrow Adipoq-lineage progenitors to osteogenesis. Thus, this article further supports Shn3 silencing as a valuable approach to treat osteopenia and accelerate fracture healing (see graphical abstract)

Abstract Wnt signaling is critically involved in fracture healing. Existing data predominantly relies on rodent models. Here, we explored local and circulating Dickkopf-1 (DKK1) levels in patients with respect to fracture healing and explore its association to sclerostin (SOST). 69 patients after surgical stabilization of long bone fractures of which six patients had impaired fracture healing were included in this study. Life-style and patient related factors with a known effect on DKK1 and SOST were recorded. DKK1 and SOST concentrations were measured using enzyme-linked immunosorbent assay (ELISA) at the fracture site and in circulation. DKK1 and SOST showed a close inverse correlation. In fracture hematoma and immediately after trauma DKK1 levels were significantly reduced while SOST levels were significantly increased, compared to healthy control. Postoperatively, DKK1 peaked at week 2 and SOST at week 8, again demonstrating a close negative correlation. Age and smoking status affected the balance of DKK1 and SOST, while type 2 diabetes and sex did not demonstrate a significant influence. Early postoperative elevation of SOST without compensatory DKK1 decrease was associated with fracture non-union in younger patients (

Abstract Introduction Fractures with delayed healing can be a serious complication, especially for athletes depending on quick return to sports. To our knowledge, no cases of increased healing of delayed union fractures with autologous biomedical interventions have been reported. Case presentation A 33-year-old Swedish professional boxer with a fractured humerus with a delayed union and a partially torn supraspinatus tendon was treated with injections of liquid platelet-rich fibrin and heat-coagulated albumin gel. He recovered almost completely from both injuries in only 1 month and could return to professional boxing in 3 months. Conclusion This case raises the hypothesis that liquid platelet-rich fibrin and heat-coagulated albumin gel may be an effective, minimally invasive intervention in fractures with a delayed union. Further research is required to test this theory.

Abstract Background Fractures heal through a process that involves angiogenesis and osteogenesis but may also lead to non-union or delayed healing. Bone marrow mesenchymal stem cells (BMSCs) have been reported to play a pivotal role in bone formation and vascular regeneration and the p75 neurotrophin receptor (p75NTR) as being an important regulator of osteogenesis. Herein, we aim to determine the potential mediation of BMSCs by p75NTR in bone healing. Methods Rat BMSCs were identified by flow cytometry (FCM) to detect cell cycle and surface markers. Then transfection of si/oe-p75NTR was performed in BMSCs, followed by Alizarin red staining to detect osteogenic differentiation of cells, immunofluorescence double staining was performed to detect the expression of p75NTR and sortilin, co-immunoprecipitation (CO-IP) was conducted to analyze the interaction between p75NTR and sortilin, and EdU staining and cell scratch assay to assess the proliferation and migration of human umbilical vein endothelial cells (HUVECs). The expression of HIF-1α, VEGF, and apoptosis-related proteins were also detected. In addition, a rat fracture healing model was constructed, and BMSCs-si-p75NTR were injected, following which the fracture condition was observed using micro-CT imaging, and the expression of platelet/endothelial cell adhesion molecule-1 (CD31) was assessed. Results The results showed that BMSCs were successfully isolated, p75NTR inhibited apoptosis and the osteogenic differentiation of BMSCs, while si-p75NTR led to a decrease in sortilin expression in BMSCs, increased proliferation and migration in HUVECs, and upregulation of HIF-1α and VEGF expression. In addition, an interaction was observed between p75NTR and sortilin. The knockdown of p75NTR was found to reduce the severity of fracture in rats and increase the expression of CD31 and osteogenesis-related proteins. Conclusion Silencing p75NTR effectively modulates BMSCs to promote osteogenic differentiation and angiogenesis, offering a novel perspective for improving fracture healing.

Background: The musculoskeletal system contains an extensive network of lymphatic vessels. Decreased lymph flow of the draining collecting lymphatics usually occurs in clinic after traumatic fractures. However, whether defects in lymphatic drainage can affect fracture healing is unclear. Methods: To investigate the effect of lymphatic dysfunction on fracture healing, we used a selective VEGFR3 tyrosine kinase inhibitor to treat tibial fractured mice for 5 weeks versus a vehicle-treated control. To ensure successfully establishing deceased lymphatic drainage model for fractured mice, we measured lymphatic clearance by near infrared indocyanine green lymphatic imaging (NIR-ICG) and the volume of the draining popliteal lymph nodes (PLNs) by ultrasound at the whole phases of fracture healing. In addition, hindlimb edema from day 0 to day 7 post-fracture, pain sensation by Hargreaves test at day 1 post-fracture, bone histomorphometry by micro-CT and callus composition by Alcian Blue-Hematoxylin/Orange G staining at day 14 post-fracture, and bone quality by biomechanical testing at day 35 post-fracture were applied to evaluate fracture healing. To promote fracture healing via increasing lymphatic drainage, we then treated fractured mice with anti-mouse podoplanin (PDPN) neutralizing antibody or isotype IgG antibody for 1 week to observe lymphatic drainage function and assess bone repair as methods described above. Results: Compared to vehicle-treated group, SAR-treatment group significantly decreased lymphatic clearance and the volume of draining PLNs. SAR-treatment group significantly increased soft tissue swelling, and reduced bone volume (BV)/tissue volume (TV), trabecular number (Tb.N), woven bone and biomechanical properties of fracture callus. In addition, anti-PDPN treated group significantly reduced the number of CD41+ platelets in PLNs and increased the number of pulsatile lymphatic vessels, lymphatic clearance and the volume of PLNs. Moreover, anti-PDPN treated group significantly reduced hindlimb edema and pain sensation and increased BV/TV, trabecular number (Tb.Th), woven bone and biomechanical properties of fracture callus. Conclusions: Inhibition of proper lymphatic drainage function delayed fracture healing. Use of a anti-PDPN neutralizing antibody reduced lymphatic platelet thrombosis (LPT), increased lymphatic drainage and improved fracture healing. The translational potential of this article: (1) We demonstrated lymphatic drainage function is crucial for fracture healing. (2) To unblock the lymphatic drainage and prevent the risk of bleeding and mortality by blood thinner, we demonstrated PDPN neutralizing antibody is a novel and safe way forward in the treatment of bone fracture healing by eliminating LPT and increasing lymphatic drainage.

Introduction: Bone healing can be improved by axial micromovement, as has been shown in animals and human patients with external fixators. In the development of smart fracture plates, the ideal amount of stroke for different fracture types in the different healing stages is currently unknown. It was hypothesized that the resulting strain in the fracture gap of a simple tibial shaft fracture does not vary with the amount of axial stroke in the plate, the fracture gap size, and the fracture angle.Methods: With finite element simulations based on body donation computed tomography data, the second invariant of the deviatoric strain tensor (J2), strain energy density, hydrostatic strain, octahedral shear strain, and percentage of the fracture gap in the “perfect healing window” were computed for different gap sizes (1–3 mm), angles (5°–60°), and plate stroke levels (0.05–0.60 mm) in three healing stages. Multiple linear regression analyses were performed.Results: Findings showed that an active fracture plate should deliver an axial stroke in the range of 0.10–0.45 mm. Different optimal stroke values were found for each healing phase, namely, 0.10–0.25 mm for the first, 0.10 mm for the second, and 0.35–0.45 mm for the third healing phase, depending on the fracture gap size and less on the fracture angle. J2, hydrostatic strain, octahedral shear strain and the strain energy density correlated with the fracture gap size and angle (all p < 0.001). The influence of the fracture gap size and angle on the variability (adjusted R2) in several outcome measures in the fracture gap was shown to vary throughout healing. The contribution to the variability of the percentage of the fracture gap in the perfect healing window was greatest during the second healing phase. For J2, strain energy density, hydrostatic strain, and octahedral shear strain, the fracture gap size showed the greatest contribution in the third fracture healing phase, while the influence of fracture angle was independent of the healing phase.Discussion: The present findings are relevant for implant development and to design clinical studies that aim to accelerate fracture healing using axial micromovement.

Abstract Ultrasonography has begun to be applied to the examination of fractures in recent years owing to its safety, noninvasiveness, portability, and high sensitivity. The subcutaneous soft tissue of children is thinner than that of adults, children's bones have a high level of elasticity and a low level of brittleness, and the pediatric fractures heal quickly and produce more callus, therefore ultrasonography is a more suitable examination in pediatric bone fractures. In this paper, we intend to review the mechanism, performance, and examination effect of ultrasound examination in bone fractures, analyze the advantages and disadvantages between ultrasound imaging and X‐ray imaging, and further propose an outlook for the application of ultrasound technology in pediatric bone fractures.

ABSTRACT Objective: Identify the predictors associated with delayed union at 6 months and non-union at 12 months in tibial shaft fractures treated with intramedullary nailing (IMN). Methods: This retrospective longitudinal study included a cohort of 218 patients who sustained tibial shaft fractures and received IMN between January 2015 and March 2022. We gathered data on a range of risk factors, including patient demographics, trauma intensity, associated injuries, fracture characteristics, soft tissue injuries, comorbidities, addictions, and treatment-specific factors. We employed logistic bivariate regression analysis to explore the factors predictive of delayed union and non-union. Results: At the 6-month follow-up, the incidence of delayed union was 28.9%. Predictors for delayed union included flap coverage, high-energy trauma, open fractures, the use of external fixation as a staged treatment, the percentage of cortical contact in simple type fractures, RUST score, and postoperative infection. After 12 months, the non-union rate was 15.6%. Conclusion: the main predictors for non-union after IMN of tibial shaft fractures are related to the trauma energy. Furthermore, the initial treatment involving external fixation and postoperative infection also correlated with non-union. Level of Evidence III; Retrospective Longitudinal Study.

BackgroundOsteoking (OK) is prescribed in traditional Chinese medicine to accelerate fracture healing. Although some studies suggest the potential efficacy of OK for fracture healing, the evidence remains inconclusive.AimTo systematically evaluate the safety of OK and its effect on fracture healing.MethodsRelevant authoritative databases were searched until 25 August 2023. Randomized controlled trials (RCTs) of patients with fractures treated with Osteoking were included. We evaluated the risk of bias using the Cochrane tool and performed a meta-analysis using the Review Manager 5.4 software package.Results13 studies involving 1123 participants were included. This meta-analysis showed that compared with observations in the control group, the OK group showed a shortened fracture healing time, increased fracture healing rate, reduced swelling regression time and ecchymosis regression time, and improved bone metabolism. In addition, the included studies did not report any serious side effects associated with the use of OK, and the mild side effects resolved without treatment.ConclusionOK therapy is beneficial and safe for accelerating fracture healing, reducing swelling, eliminating ecchymosis, and improving bone metabolism. However, the meta-analysis results do not support OK treatment for improving the fracture healing rate at all fracture sites and reducing pain across all fracture sites. Further original, high-quality studies are needed to validate these findings.Systematic Review Registration:https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=452430, identifier CRD42023452430.

Approximately half of bone fractures that do not heal properly (non-union) can be accounted to insufficient angiogenesis. The processes of angiogenesis and osteogenesis are spatiotemporally regulated in the complex process of fracture healing that requires a substantial amount of energy. It is thought that a metabolic coupling between angiogenesis and osteogenesis is essential for successful healing. However, how this coupling is achieved remains to be largely elucidated. Here, we will discuss the most recent evidence from literature pointing towards a metabolic coupling between angiogenesis and osteogenesis. We will describe the metabolic profiles of the cell types involved during fracture healing as well as secreted products in the bone microenvironment (such as lactate and nitric oxide) as possible key players in this metabolic crosstalk.

Abstract Purpose Previous studies have shown that DNA methyltransferase 3b (Dnmt3b) is the only Dnmt responsive to fracture repair and Dnmt3b ablation in Prx1-positive stem cells and chondrocyte cells both delayed fracture repair. Our study aims to explore the influence of Dnmt3b ablation in Gli1-positive stem cells in fracture healing mice and the underlying mechanism. Methods We generated Gli1-CreERT2; Dnmt3bflox/flox (Dnmt3b Gli1ER ) mice to operated tibia fracture. Fracture callus tissues of Dnmt3b Gli1ER mice and control mice were collected and analyzed by X-ray, micro-CT, biomechanical testing, histopathology and TUNEL assay. Results The cartilaginous callus significantly decrease in ablation of Dnmt3b in Gli1-positive stem cells during fracture repair. The chondrogenic and osteogenic indicators (Sox9 and Runx2) in the fracture healing tissues in Dnmt3b Gli1ER mice much less than control mice. Dnmt3b Gli1ER mice led to delayed bone callus remodeling and decreased biomechanical properties of the newly formed bone during fracture repair. Both the expressions of Caspase-3 and Caspase-8 were upregulated in Dnmt3b Gli1ER mice as well as the expressions of BCL-2. Conclusions Our study provides an evidence that Dnmt3b ablation Gli1-positive stem cells can affect fracture healing and lead to poor fracture healing by regulating apoptosis to decrease chondrocyte hypertrophic maturation.

Aims: Despite limited clinical scientific backing, an additional trochanteric stabilizing plate (TSP) has been advocated when treating unstable trochanteric fractures with a sliding hip screw (SHS). We aimed to explore whether the TSP would result in less post operative fracture motion, compared to SHS alone. Methods: Overall, 31 patients with AO/OTA 31-A2 trochanteric fractures were randomized to either a SHS alone or a SHS with an additional TSP. To compare postoperative fracture motion, radiostereometric analysis (RSA) was performed before and after weightbearing, and then at four, eight, 12, 26, and 52 weeks. With the “after weightbearing” images as baseline, we calculated translations and rotations, including shortening and medialization of the femoral shaft. Results: Similar migration profiles were observed in all directions during the course of healing. At one year, eight patients in the SHS group and 12 patients in the TSP group were available for analysis, finding a clinically non-relevant, and statistically non-significant, difference in total translation of 1 mm (95% confidence interval -4.7 to 2.9) in favour of the TSP group. In line with the migration data, no significant differences in clinical outcomes were found. Conclusion: The TSP did not influence the course of healing or postoperative fracture motion compared to SHS alone. Based on our results, routine use of the TSP in AO/OTA 31-A2 trochanteric fractures cannot be recommended. The TSP has been shown, in biomechanical studies, to increase stability in sliding hip screw constructs in both unstable and intermediate stable trochanteric fractures, but the clinical evidence is limited. This study showed no advantage of the TSP in unstable (AO 31-A2) fractures in elderly patients when fracture movement was evaluated with radiostereometric analysis. Cite this article: Bone Jt Open 2024;5(1):37–45.

Blake M Bacevich,1 Richard David James Smith,1 Alec M Reihl,1 Augustus D Mazzocca,1,2 Ian D Hutchinson1 1Division of Sports Medicine, Department of Orthopaedic Surgery, Massachusetts General Hospital, Harvard Medical School, Massachusetts General Brigham, Boston, MA, USA; 2Medical Director, Division of Sports Medicine, Department of Orthopaedic Surgery, Massachusetts General Brigham, Boston, MA, USACorrespondence: Ian D Hutchinson, Division of Sports Medicine, Department of Orthopaedic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, Tel +1 781-487-6166, Fax +1 781-487-6826, Email ihutchinson@mgh.harvard.eduAbstract: Despite significant advances in the understanding and delivery of osteosynthesis, fracture non-union remains a challenging clinical problem in orthopaedic surgery. To bridge the gap, basic science characterization of fracture healing provides a platform to identify and target biological strategies to enhance fracture healing. Of immense interest, Platelet-rich plasma (PRP) is a point of care orthobiologic that has been extensively studied in bone and soft tissue healing given its relative ease of translation from the benchtop to the clinic. The aim of this narrative review is to describe and relate pre-clinical in-vitro and in-vivo findings to clinical observations investigating the efficacy of PRP to enhance bone healing for primary fracture management and non-union treatment. A particular emphasis is placed on the heterogeneity of PRP preparation techniques, composition, activation strategies, and delivery. In the context of existing data, the routine use of PRP to enhance primary fracture healing and non-union management cannot be supported. However, it is acknowledged that extensive heterogeneity of PRP treatments in clinical studies adds obscurity; ultimately, refinement (and consensus) of PRP treatments for specific clinical indications, including repetition studies are warranted.Keywords: platelet rich plasma, bone regeneration, fracture healing, fractures, ununited

Abstract The senescence-associated secretory phenotype (SASP) is a generic term for the secretion of cytokines, such as pro-inflammatory factors and proteases. It is a crucial feature of senescent cells. SASP factors induce tissue remodeling and immune cell recruitment. Previous studies have focused on the beneficial role of SASP during embryonic development, wound healing, tissue healing in general, immunoregulation properties, and cancer. However, some recent studies have identified several negative effects of SASP on fracture healing. Senolytics is a drug that selectively eliminates senescent cells. Senolytics can inhibit the function of senescent cells and SASP, which has been found to have positive effects on a variety of aging-related diseases. At the same time, recent data suggest that removing senescent cells may promote fracture healing. Here, we reviewed the latest research progress about SASP and illustrated the inflammatory response and the influence of SASP on fracture healing. This review aims to understand the role of SASP in fracture healing, aiming to provide an important clinical prevention and treatment strategy for fracture. Clinical trials of some senolytics agents are underway and are expected to clarify the effectiveness of their targeted therapy in the clinic in the future. Meanwhile, the adverse effects of this treatment method still need further study.

Objectives Scaphoid nonunion remains a challenging problem to manage with no general consensus on its treatment recommendations. We propose a novel minimally invasive (MIS) technique of arthroscopic bone grafting (ABG) with robot‐assisted fixation for the treatment of scaphoid nonunions. Methods Patients with radiographically proven scaphoid nonunion treated by this novel surgical technique were included. Following arthroscopic debridement and iliac crest bone grafting, the scaphoid was fixed percutaneously using either multiple Kirschner (K)‐wires or a headless compression screw using a robotic navigation system. Results Six male patients with an average age of 29.2 years were enrolled. Four patients had scaphoid waist fractures, and the other two were proximal pole fractures. During wrist arthroscopy, punctate bleeding of the proximal scaphoid fragment was observed in four out of the six patients. Half of the patients were fixed using a headless compression screw and the other half using multiple K‐wires. All the guidewires were placed with a single‐attempt using the robotic navigation system. Postoperatively, all the scaphoid fractures had complete radiographic union by 16 weeks. At a mean follow‐up of 18.3 months, there were significant improvements in wrist range of motion, grip strength, and patient‐rated outcomes. No intraoperative or early postoperative complications were encountered in any of our patients. Conclusion Arthroscopic bone grafting with robot‐assisted fixation is a feasible and promising therapeutic option for scaphoid nonunions, regardless of the vascularity of the proximal pole fragment. This novel technique allows for anatomic restoration of the scaphoid alignment and accurate, targeted placement of implants into the scaphoid nonunion site within a single‐attempt using a robotic navigation system.

The bone fracture cases have been increasing yearly, accompanied by the increased number of patients experiencing non-union or delayed union after their bone fracture. Although clinical materials facilitate fracture healing (e.g., metallic and composite materials), they cannot fulfill the requirements due to the slow degradation rate, limited osteogenic activity, inadequate osseointegration ability, and suboptimal mechanical properties. Since early 2000, nanomaterials successfully mimic the nanoscale features of bones and offer unique properties, receiving extensive attention. This paper reviews the achievements of nanomaterials in treating bone fracture (e.g., the intrinsic properties of nanomaterials, nanomaterials for bone defect filling, and nanoscale drug delivery systems in treating fracture delayed union). Furthermore, we discuss the perspectives on the challenges and future directions of developing nanomaterials to accelerate fracture healing.

This study aimed to evaluate the osteogenic potential of mesenchymal stromal cell (MSC) spheroids combined with the basic fibroblast growth factor (bFGF) in a mouse femur fracture model. To begin, MSC spheroids were generated, and the expression of key trophic factors (bFGF Bmp2, and Vegfa) was assessed using quantitative PCR (qPCR). A binding assay confirmed the interaction between the bFGF and the spheroids’ extracellular matrix. The spheroid cultures significantly upregulated bFGF, Bmp2, and Vegfa expression compared to the monolayers (p < 0.001), and the binding assay demonstrated effective bFGF binding to the MSC spheroids. Following these in vitro assessments, the mice were divided into five groups for the in vivo study: (1) no treatment (control), (2) spheroids alone, (3) bFGF alone, (4) bFGF-loaded spheroids (bFGF-spheroids), and (5) non-viable (frozen) bFGF-loaded spheroids (bFGF-dSpheroids). Bone formation was analyzed by a micro-CT, measuring the bone volume (BV) and bone mineral content (BMC) of the mice four weeks post-fracture. A high dose of the bFGF (10 µg) significantly promoted bone formation regardless of the presence of spheroids, as evidenced by the increases in BV (bFGF, p = 0.010; bFGF-spheroids, p = 0.006; bFGF-dSpheroids, p = 0.032) and BMC (bFGF, p = 0.023; bFGF-spheroids, p = 0.004; bFGF-dSpheroids, p = 0.014), compared to the controls. In contrast, a low dose of the bFGF (1 µg) combined with the MSC spheroids significantly increased BV and BMC compared to the control (BV, p = 0.012; BMC, p = 0.015), bFGF alone (BV, p = 0.012; BMC, p = 0.008), and spheroid (BV, p < 0.001; BMC, p < 0.001) groups. A low dose of the bFGF alone did not significantly promote bone formation (p > 0.05). The non-viable (frozen) spheroids loaded with a low dose of the bFGF resulted in a higher BV and BMC compared to the spheroids alone (BV, p = 0.003; BMC, p = 0.017), though the effect was less pronounced than in the viable spheroids. These findings demonstrate the synergistic effect of the bFGF and MSC spheroids on bone regeneration. The increased expression of the BMP-2 and VEGF observed in the initial experiments, coupled with the enhanced bone formation in vivo, highlight the therapeutic potential of this combination. Future studies will aim to elucidate the underlying molecular mechanisms and assess the long-term outcomes for bone repair strategies.

Background: Electromagnetic field therapy is gaining attention for its potential in treating bone disorders, with Extracorporeal Magnetotransduction Therapy (EMTT) emerging as an innovative approach. EMTT offers a higher oscillation frequency and magnetic field strength compared to traditional Pulsed Electromagnetic Field (PEMF) therapy, showing promise in enhancing fracture healing and non-union recovery. However, the mechanisms underlying these effects remain unclear. Results: This study demonstrates that EMTT significantly enhances osteoblast bone formation at multiple levels, from gene expression to extracellular matrix mineralization. Key osteoblastogenesis regulators, including SP7 and RUNX2, and bone-related genes such as COL1A1, ALPL, and BGLAP, were upregulated, with expression levels surpassing those of the control group by over sevenfold (p < 0.001). Enhanced collagen synthesis and mineralization were confirmed by von Kossa and Alizarin Red staining, indicating increased calcium and phosphate deposition. Additionally, calcium imaging revealed heightened calcium influx, suggesting a cellular mechanism for EMTT’s osteogenic effects. Importantly, EMTT did not compromise cell viability, as confirmed by live/dead staining and WST-1 assays. Conclusion: This study is the first to show that EMTT can enhance all phases of osteoblastogenesis and improve the production of critical mineralization components, offering potential clinical applications in accelerating fracture healing, treating osteonecrosis, and enhancing implant osseointegration.

Delayed union and non-union of fractures continue to be a major problem in trauma and orthopedic surgery. These cases are challenging for the surgeon. In addition, these patients suffer from multiple surgeries, pain and disability. Furthermore, these cases are a major burden on healthcare systems. The scientific community widely agrees that the stability of fixation plays a crucial role in determining the outcome of osteosynthesis. The extent of stabilization affects factors like fracture gap strain and fluid flow, which, in turn, influence the regenerative processes positively or negatively. Nonetheless, a growing body of literature suggests that during the fracture healing process, there exists a critical time frame where intervention can stimulate the bone's return to its original form and function. This article provides a summary of existing evidence in the literature regarding the impact of different levels of fixation stability on the strain experienced by newly forming tissues. We will also discuss the timing and nature of this “window of opportunity” and explore how current knowledge is driving the development of new technologies with design enhancements rooted in mechanobiological principles.

The elderly exhibit a reduced healing capacity after fracture, which is often associated with delayed or failed bone healing. This is due to a plethora of factors, such as an impaired bone vascular system and delayed angiogenesis. The phosphodiesterase-5 (PDE-5) inhibitor sildenafil exerts pro-angiogenic and pro-osteogenic effects. Hence, we herein investigated in aged mice whether sildenafil can improve fracture healing. For this purpose, 40 aged CD-1 mice (16–18 months) were daily treated with 5 mg/kg body weight sildenafil (n = 20) or vehicle (control, n = 20) by oral gavage. The callus tissue of their femora was analyzed at 2 and 5 weeks after fracture by X-ray, biomechanics, micro-computed tomography (µCT), histology, immunohistochemistry as well as Western blotting. These analyses revealed a significantly increased bone volume and higher ratio of callus to femoral bone diameter in sildenafil-treated mice at 5 weeks after fracture when compared to controls. This was associated with a reduced number and activity of osteoclasts at 2 weeks after fracture, most likely caused by an increased expression of osteoprotegerin (OPG). Taken together, these findings indicate that sildenafil does not improve fracture healing in the elderly but delays the process of bone remodeling most likely by reducing the number and activity of osteoclasts within the callus tissue.

Abstract Background Non-union formation still represents a major burden in trauma and orthopedic surgery. Moreover, aged patients are at an increased risk for bone healing failure. Parathyroid hormone (PTH) has been shown to accelerate fracture healing in young adult animals. However, there is no information whether PTH also stimulates bone regeneration in atrophic non-unions in the aged. Therefore, the aim of the present study was to analyze the effect of PTH on bone regeneration in an atrophic non-union model in aged CD-1 mice. Methods After creation of a 1.8 mm segmental defect, mice femora were stabilized by pin-clip fixation. The animals were treated daily with either 200 μg/kg body weight PTH 1–34 (n = 17) or saline (control; n = 17) subcutaneously. Bone regeneration was analyzed by means of X-ray, biomechanics, micro-computed tomography (µCT) imaging as well as histological, immunohistochemical and Western blot analyses. Results In PTH-treated animals bone formation was markedly improved when compared to controls. This was associated with an increased bending stiffness as well as a higher number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and CD31-positive microvessels within the callus tissue. Furthermore, PTH-treated aged animals showed a decreased inflammatory response, characterized by a lower number of MPO-positive granulocytes and CD68-positive macrophages within the bone defects when compared to controls. Additional Western blot analyses demonstrated a significantly higher expression of cyclooxygenase (COX)-2 and phosphoinositide 3-kinase (PI3K) in PTH-treated mice. Conclusion Taken together, these findings indicate that PTH is an effective pharmacological compound for the treatment of non-union formation in aged animals.

Abstract Background Fractures are the most common orthopedic diseases. It is known that static magnetic fields (SMFs) can contribute to the maintenance of bone health. However, the effect and mechanism of SMFs on fracture is still unclear. This study is aim to investigate the effect of moderate static magnetic fields (MMFs) on bone structure and metabolism during fracture healing. Methods Eight-week-old male C57BL/6J mice were subjected to a unilateral open transverse tibial fracture, and following treatment under geomagnetic field (GMF) or MMF. The micro-computed tomography (Micro-CT) and three-point bending were employed to evaluate the microarchitecture and mechanical properties. Endochondral ossification and bone remodeling were evaluated by bone histomorphometric and serum biochemical assay. In addition, the atomic absorption spectroscopy and ELISA were utilized to examine the influence of MMF exposure on iron metabolism in mice. Results MMF exposure increased bone mineral density (BMD), bone volume per tissue volume (BV/TV), mechanical properties, and proportion of mineralized bone matrix of the callus during fracture healing. MMF exposure reduced the proportion of cartilage in the callus area during fracture healing. Meanwhile, MMF exposure increased the number of osteoblasts in callus on the 14th day, and reduced the number of osteoclasts on the 28th day of fracture healing. Furthermore, MMF exposure increased PINP and OCN levels, and reduced the TRAP-5b and β-CTX levels in serum. It was also observed that MMF exposure reduced the iron content in the liver and callus, as well as serum ferritin levels while elevating the serum hepcidin concentration. Conclusions MMF exposure could accelerate fracture healing via promote the endochondral ossification and bone formation while regulating systemic iron metabolism during fracture healing. This study suggests that MMF may have the potential to become a form of physical therapy for fractures.

Aims: Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy. Methods: Zucker rats, WT and homozygous for leptin receptor mutation (ZDF), were fed a moderately high-fat diet to induce T2DM only in the ZDF animals. At ten weeks of age, open femoral fractures were simulated using a unilateral osteotomy stabilized with an external fixator. At three weeks post-surgery, the fractured femur from each animal was retrieved for analysis. Callus formation and the extent of healing were assessed by radiograph and histology. Bone tissue was processed for total RNA extraction and messenger RNA (mRNA) sequencing (mRNA-Seq). Results: Radiographs and histology demonstrated impaired fracture healing in ZDF rats with incomplete bony bridge formation and an influx of intramedullary inflammatory tissue. In comparison, near-complete bridging between cortices was observed in Sham WT animals. Of 13,160 genes, mRNA-Seq analysis identified 13 that were differentially expressed in ZDF rat callus, using a false discovery rate (FDR) threshold of 10%. Seven genes were upregulated with high confidence (FDR = 0.05) in ZDF fracture callus, most with known roles in inflammation. Conclusion: These findings suggest that elevated or prolonged inflammation contributes to delayed fracture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients. Cite this article: Bone Joint Res 2023;12(10):657–666.

Abstract Objective We aim to describe an experimental model for studying femoral fractures in rats after exposure to ionizing radiation, demonstrating a way to apply a substance for analysis, the method for patterning fracture and irradiation, and how to evaluate its effectiveness based on radiographic studies. Methods We used 24 rats divided into 2 groups of 12 animals each. The STUDY group was exposed to ionizing radiation and treated with saline solution, and the CONTROL group was not exposed to radiation and was treated with saline solution. All animals were subjected to standardized fracture of the right femur that was fixed with intramedullary wire. The efficiency of the bone union was assessed by radiographic exam. Results Fracture healing was more efficient in bones not exposed to ionizing radiation (p = 0.012). All fractures met the criteria of being simple, diaphyseal, transverse or short oblique. Conclusion The experimental model presented is an efficient alternative for the study of fractures in irradiated bones in rats.

Abstract Background Treatment of distal tibial fractures is a challenge due to their specific anatomical location. However, there is no appropriate mouse model to simulate a clinical distal tibial fracture for basic research. The aim of this investigation was to evaluate the feasibility of simulating a clinical fracture of the distal tibia of mice and to investigate the effect of ovariectomy (OVX)-induced osteoporosis on fracture healing in this model. Methods Sixty female 8-week-old C57BL/6 mice were randomly divided into two groups, either sham or OVX. A semi-fixation distal tibia fracture was established in the right tibia after 8 weeks of OVX. The right tibias were collected at 7, 14, 21, and 28 days post fracture. Results In the semi-fixation distal tibia fracture model, the posterior callus in the sham group showed excessive bone resorption and lower bone mass phenotype compared with the anterior site; a similar trend was not found in the OVX group. At 28 days post fracture, the posterior callus was more mineralized than the anterior callus in the OVX group. Although the fracture healing of the sham group showed a special phenotype in this mode, the progress and quality of fracture healing were still better than those of the OVX group. Conclusion A semi-fixed distal tibial closed fracture mouse model was successfully established. In this model, excess bone resorption of the posterior callus impaired normal fracture healing, but not in OVX-induced osteoporotic bone. Although the stress shielding effect was not observed in the OVX group, impaired bone healing caused by OVX was still present. Our results suggest that this fracture model may have potential for studies on distal tibial fractures and stress shielding.

Objective This is a rare study comparing the radiological and functional outcomes of fixation after reduction with in situ fixation group using Femoral Neck System (FNS). The aim of this study was to introduce a simple, innovative, minimally invasive method to reduce valgus‐impacted and tilted femoral neck fractures without soft tissue or cartilage injury. Methods A retrospective comparative analysis of 46 patients between May 2020 and February 2022 was performed. In the control group, 23 patients underwent in situ fixation without reduction. In the study group, another 23 patients were managed by fixation after reduction using a percutaneous pull‐out technique with a full threaded Steinmann pin. Caput‐collum‐diaphysis (CCD) angle, tilt, and femoral neck shortenings were compared between the two groups. In addition, Harris Hip Score (HHS) was evaluated and compared at 1 year after surgery. Basically, independent samples t‐test was used to compare radiological and functional results. Results Patients' initial valgus and tilt angles were not significantly different between the groups (n.s.). However, the CCD and tilt angles measured immediately and at one year postoperatively were significantly different between the groups (p

This study aimed to find out how estrogen affects fracture healing. Thirty adult bitches were employed, and they were put into three groups at random: First, second, and third. A mixture of ketamine and xylazine has been used to anesthetize experimental animals. Electrical drilling in the distal part of the tibial bone with a 10 mm diameter was done on the first group to create a hole in the tibia. The Tibia hole was created after ovariectomy in the second group but after ovariohysterectomy in the third group. Clinical, estrogen assay and histopathological characteristics were assessed in all groups. During the first week after surgery, clinical results showed that symptoms of inflammation in the surgical region were substantial in the first group, while the inflammation symptoms in the second group were similar to those in the first group; however, it was milder in the third group. In the second group, estrogen levels reduced modestly; however, in the third group, estrogen levels declined significantly in the fourth week after surgery. According to histopathological findings, the proliferation of osteoclasts and osteoblasts at 15 and 30 days after surgery is much more pronounced in the first and second groups than in the third groups. Osteocytes are also more common in the first group than in the second and third groups. According to the results of this study, fracture healing in the third group is much less quantitative and qualitative than in the other groups.

Abstract Non-union formation represents a major complication in trauma and orthopedic surgery. The phosphodiesterase-5 (PDE-5) inhibitor sildenafil has been shown to exert pro-angiogenic and pro-osteogenic effects in vitro and in vivo. Therefore, the aim of the present study was to analyze the impact of sildenafil in an atrophic non-union model in mice. After creation of a 1.8 mm segmental defect, mice femora were stabilized by pin-clip fixation. Bone regeneration was analyzed by means of X-ray, biomechanics, photoacoustic and micro-computed tomography (µCT) imaging as well as histological, immunohistochemical and Western blot analyses at 2, 5 and 10 weeks after surgery. The animals were treated daily with either 5 mg/kg body weight sildenafil (n = 35) or saline (control; n = 35) per os. Bone formation was markedly improved in defects of sildenafil-treated mice when compared to controls. This was associated with a higher bending stiffness as well as an increased number of CD31-positive microvessels and a higher oxygen saturation within the callus tissue. Moreover, the bone defects of sildenafil-treated animals contained more tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and CD68-positive macrophages and exhibited a higher expression of the pro-angiogenic and pro-osteogenic markers cysteine rich protein (CYR)61 and vascular endothelial growth factor (VEGF) when compared to controls. These findings demonstrate that sildenafil acts as a potent stimulator of angiogenesis and bone regeneration in atrophic non-unions.