Unique Spatial Transcriptomic Profiling of the Murine Femoral Fracture Callus: A Preliminary Report.

Fracture callus formation is a dynamic stage of bone activity and repair with precise, spatially localized gene expression. Metastatic breast cancer impairs fracture healing by disrupting bone homeostasis and imparting an altered genomic profile. Previous sequencing techniques such as single-cell RNA and in situ hybridization are limited by missing spatial context and low throughput, respectively. We present a preliminary approach using the Visium CytAssist spatial transcriptomics platform to provide the first spatially intact characterization of genetic expression changes within an orthopedic model of impaired fracture healing. Tissue slides prepared from BALB/c mice with or without MDA-MB-231 metastatic breast cancer cells were used. Both unsupervised clustering and histology-based annotations were performed to identify the hard callus, soft callus, and interzone for differential gene expression between the wild-type and pathological fracture model. The spatial transcriptomics platform successfully localized validated genes of the hard (Dmp1, Sost) and soft callus (Acan, Col2a1). The fibrous interzone was identified as a region of extensive genomic heterogeneity. MDA-MB-231 samples demonstrated downregulation of the critical bone matrix and structural regulators that may explain the weakened bone structure of pathological fractures. Spatial transcriptomics may represent a valuable tool in orthopedic research by providing temporal and spatial context.

Effects of Focal Cerebellar Injury on Fracture Healing and Oxidative Stress in Rat Model: An Experimental Animal Study.

AIM: To examine the effect of cerebellar damage on the process of fracture healing. MATERIAL AND METHODS: A total of forty-two male rats were selected at random and subsequently allocated into three distinct groups. The experimentals were divided into two subgroups within each group, with the intention of sacrificing them during the third and sixth weeks. Group 1 had isolated femoral fracture, Group 2 had femoral fracture after craniotomy, and Group 3 had femoral fracture accompanying cerebellar injury after craniotomy. Left femoral fractures in rats in all groups were treated using an intramedullary Kirschner wire. Radiological, histological, and biochemical evaluations were conducted at 3 and 6 weeks to assess the processes of fracture healing. To determine the effects of fracture healing and cerebellar injury on oxidant-antioxidant systems, catalase (CAT), malondialdehyde, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were measured. RESULTS: Between the time frame of 3 to 6 weeks, Group 3 had higher radiography scores, alkaline phosphatase levels, callus/ diaphyse ratio, callus improvement, and bone mineral density in comparison to the other groups. The activity of SOD was found to be statistically negligible in all groups, suggesting that SOD does not have a substantial impact on fracture healing in cerebellar injury. However, notable increases in the activity of GPx and CAT enzymes were observed, showing their considerable involvement in the process of fracture healing. CONCLUSION: Cerebellar injury reduces the oxidative stress in the fracture area and contributes positively to fracture healing by means of radiologically, biochemically and histopathologically.

Tenascin-C promotes endochondral ossification and fracture healing through Hedgehog and Hippo signaling.

Fractures are frequent and severe musculoskeletal injuries. This study aimed to investigate the function of tenascin-C (TNC) in regulating chondrogenic during fracture healing and elucidate the underlying molecular mechanisms. A well-established femur fracture model in male C57BL/6J mice was used to transect the middle diaphysis of the femur. To identify the essential role of TNC, shTNC lentiviruses or TNC protein were administered in the animal model. Micro-CT analysis, histologic analysis, immunostaining assays, and gene expression analysis were employed to investigate the effect of TNC during fracture healing. An in vitro mesenchymal stem cell culture system was developed to investigate the role and molecular mechanism of TNC in regulating chondrogenesis. TNC expression was induced at the inflammatory phase and peaked at the cartilaginous callus phase during fracture healing. Knockdown of TNC expression in callus results in decreased callus formation and impaired fracture healing. Conversely, administration of exogenous TNC promoted chondrogenic differentiation, cartilage template formation and ultimately improved fracture healing. Both the Hedgehog and Hippo signaling pathways were found to be involved in the pro-chondrogenic function of TNC. Our observations demonstrate that TNC is a crucial factor responsible for endochondral ossification in fracture healing and provide a potential therapeutic strategy for promoting fracture healing.

The influence of femoral lytic tumors segmentation on autonomous finite element analysis.

BACKGROUND: The validated CT-based autonomous finite element system Simfini (Yosibash et al., 2020) is used in clinical practice to assist orthopedic oncologists in determining the risk of pathological femoral fractures due to metastatic tumors. The finite element models are created automatically from CT-scans, assigning to lytic tumors a relatively low stiffness as if these were a low-density bone tissue because the tumors could not be automatically identified. METHODS: The newly developed automatic deep learning algorithm which segments lytic tumors in femurs, presented in (Rachmil et al., 2023), was integrated into Simfini. Finite element models of twenty femurs from ten CT-scans of patients with femoral lytic tumors were analyzed three times using: the original methodology without tumor segmentation, manual segmentation of the lytic tumors, and the new automatic segmentation deep learning algorithm to identify lytic tumors. The influence of explicitly incorporating tumors in the autonomous finite element analysis on computed principal strains is quantified. These serve as an indicator of femoral fracture and are therefore of clinical significance. FINDINGS: Autonomous finite element models with segmented lytic tumors had generally larger strains in regions affected by the tumor. The deep learning and manual segmentation of tumors resulted in similar average principal strains in 19 regions out of the 23 regions within 15 femurs with lytic tumors. A high dice similarity score of the automatic deep learning tumor segmentation did not necessarily correspond to minor differences compared to manual segmentation. INTERPRETATION: Automatic tumor segmentation by deep learning allows their incorporation into an autonomous finite element system, resulting generally in elevated averaged principal strains that may better predict pathological femoral fractures.

Is there genetic susceptibility for atypical femoral fractures?

The mechanisms underlying AFF remain unclear, with hypotheses including bone turnover suppression and morphological variation. Recent studies have suggested a potential genetic susceptibility to AFF. A scoping review was conducted using PubMed to identify studies published since 2016. Twenty-one studies were identified, focusing on histological and genetic analysis of AFF patients and Bisphosphonates users. Biopsies and imaging modalities were used to assess histological and morphometric parameters, while genetic sequencing was performed to identify variants in target genes. Genetic studies identified variants in geranylgeranyl diphosphate synthase 1 (GGPS1) and CYP1A1 genes, which play roles in osteoclast function and drug metabolism, respectively. Functional analysis revealed reduced enzymatic activity in mutant variants of these genes, which could be further inhibited by BP use. Other genes, such as ATRAID, ALPL, and COL1A2, were also associated with AFF. Histomorphometric studies supported the hypothesis of bone turnover suppression in AFF, with alterations in tissue mechanical properties and microarchitecture observed, particularly in cortical bone. The findings suggest a potential genetic susceptibility to AFF, with variants in GGPS1 and CYP1A1 genes affecting osteoblast and osteoclast function. Bone turnover suppression and altered tissue properties contribute to the pathogenesis of AFF.

MiR-22-3p facilitates bone marrow mesenchymal stem cell osteogenesis and fracture healing through the SOSTDC1-PI3K/AKT pathway.

Bone fractures are the most common form of musculoskeletal trauma worldwide. Numerous microRNAs (miRNAs) have been suggested to be participants in regulating bone-related diseases. Recent studies revealed the regulatory role of miR-22-3p in osteogenic differentiation, but its role in fracture healing has not been investigated previously. Here, a rat femoral fracture model was established, Bone marrow mesenchymal stem cells (BMSCs) were isolated to detect the specific function and underlying mechanisms of miR-22-3p. MiR-22-3p and sclerostin domain-containing 1 (SOSTDC1) expression was determined by RT-qPCR and immunohistochemistry staining. The levels of proteins associated with osteogenic differentiation were assessed by western blotting. Flow cytometry was conducted to identify the isolated rat BMSCs. Alizarin red staining, alkaline phosphatase staining and Oil Red O staining were used to evaluate the osteogenic and adipogenic differentiation of rat BMSCs. The interaction between miR-22-3p and SOSTDC1 was verified using a luciferase reporter assay. Haematoxylin and Eosin (H&E) staining of the bone tissues was performed to analyse the effect of miR-22-3p on histopathological changes in vivo. MiR-22-3p was downregulated in the callus tissues of rat femoral fracture, while the expression of SOSTDC1 was upregulated. The isolated rat BMSCs had the capacity for both osteogenic and adipogenic differentiation. The differentiation capacity of BMSCs into osteoblasts was increased by miR-22-3p overexpression. MiR-22-3p activated the PI3K/AKT pathway by targeting SOSTDC1. SOSTDC1 overexpression and PI3K/AKT signalling inhibitor LY294002 abolished the enhancing effect of miR-22-3p overexpression on the osteogenesis of BMSCs. Thus MiR-22-3p facilitated the femoral fracture healing in rats. MiR-22-3p overexpression promoted fracture healing via the activation of PI3K/AKT pathway by targeting SOSTDC1.

Femoral metastatic pathological fractures, impending and actual fractures - A patient survival study.

INTRODUCTION: The skeleton is a common site for metastases. Prostate, breast, lung, renal and thyroid carcinomas account for 80 % of the original cancers, with the femur being the most affected long bone. With improved oncological treatments, prolonged patient survival leads to an increased prevalence of osseous metastases. This study examines the impact of preventive surgery for impending femoral pathological fracture (IFF), versus treatment of pathological femur fracture (PFF) on patient mortality and morbidity. METHODS: Retrospective cohort of 174 patients undergoing surgery due to femoral metastases (2004-2015). Eighty-two patients were with PFF, and 92 were with IFF based on the Mirels' score. The followed-up period was until 2016. Demographic data, oncological, pathological, radiation, surgical reports, outpatient clinical records, and imaging studies were examined. Exclusion criteria included primary tumours and Multiple Myeloma. RESULTS: The mean age was 64.8 ± 13.3 and 60.2 ± 11.9 years (p = 0.02) in the PFF and the IFF cohorts, with 62.1 % women and 57 % men. The breast was the most common source of femoral metastases. The average Mirels' score was 10 ± 1.2. There was an association between tumour origin and survival. Carcinoma of the lung had the worst survival, while the prostate had the most prolonged survival. Survival rates differed between IFF and PFF (p = 0.03). Postoperative complications occurred in 26 % of the patient, with no difference between IFF & PFF. CONCLUSION: Breast and lung are the most common tumours to metastasize the femur. Our study revalidates that pathological femoral fractures impede patient survival compared to impending fractures and should undergo preventive surgery. Postoperative complications do not differ between IFF and PFF but remain relatively high. Overall, patients with proximal femoral metastatic disease survive longer than previously published, probably due to improved treatment modalities.

Diclofenac, a NSAID, delays fracture healing in aged mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, belong to the most prescribed analgesic medication after traumatic injuries. However, there is accumulating evidence that NSAIDs impair fracture healing. Because bone regeneration in aged patients is subject to significant changes in cell differentiation and proliferation as well as a markedly altered pharmacological action of drugs, we herein analyzed the effects of diclofenac on bone healing in aged mice using a stable closed femoral facture model. Thirty-three mice (male n = 14, female n = 19) received a daily intraperitoneal injection of diclofenac (5 mg/kg body weight). Vehicle-treated mice (n = 29; male n = 13, female n = 16) served as controls. Fractured mice femora were analyzed by means of X-ray, biomechanics, micro computed tomography (µCT), histology and Western blotting. Biomechanical analyses revealed a significantly reduced bending stiffness in diclofenac-treated animals at 5 weeks after fracture when compared to vehicle-treated controls. Moreover, the callus tissue in diclofenac-treated aged animals exhibited a significantly reduced amount of bone tissue and higher amounts of fibrous tissue. Further histological analyses demonstrated less lamellar bone after diclofenac treatment, indicating a delay in callus remodeling. This was associated with a decreased number of osteoclasts and an increased expression of osteoprotegerin (OPG) during the early phase of fracture healing. These findings indicate that diclofenac delays fracture healing in aged mice by affecting osteogenic growth factor expression and bone formation as well as osteoclast activity and callus remodeling.

The effect of pre-fracture proximal femur geometry on hip fracture type in elderly patients.

This study aimed to analyze the relationship between fracture type by determining data on the geometry of the proximal femur in the pre-fracture period in patients over 65 years of age who had hip fractures as a result of low-energy trauma. A total of 127 patients who were admitted to the hospital for reasons other than hip pathology within 1 year before the occurrence of hip fracture and who had an anterior-posterior pelvic X-ray were included in the study. Measurements were made to evaluate the proximal femur geometry, neck shaft angle, central edge angle, femoral head diameter, femoral neck diameter, femoral neck length, femoral offset length, femoral neck axial length, hip axis length, and femoral shaft diameter. As a result of these measurements, analyses were performed to determine the relationship between the control group and fracture types. The mean Neck shaft angle scores were significantly higher in both fracture types than in the control group (P = .034, P = .002). The mean Femoral offset length values of both fracture types were lower than those of the control group (P = .002, P = .011, respectively). Multiple logistic regression analysis revealed that the risk of collum femoris fracture increased as the Femoral head diameter value increased. (OD = 0.21, P = .002). The geometric parameters of the proximal femur play an important role in the formation of hip fracture types. Therefore, differences in proximal femur geometry in hip fracture types should be considered, and patient-focused choices should be made regarding the surgical procedures and implants to be used during fracture fixation.

Intramedullary nailing versus plate compound osteosynthesis in subtrochanteric and diaphyseal pathologic femoral fractures: a retrospective cohort study.

PURPOSE: Pathologic fractures of the extremities due to carcinoma metastases require individual and patient prognosis-related stabilization procedures. Quick remobilization of the patient to restore the quality of life is of high importance, especially in the case of subtrochanteric and diaphyseal femoral fractures. In our retrospective cohort study, we evaluated intraoperative blood loss, length of operation, complication rate, and regain of lower extremity function in plate compound osteosynthesis (PCO) versus intramedullary nailing (IM) for subtrochanteric and diaphyseal pathologic fractures of the femur. METHODS: Between January 2010 and July 2021, we retrospectively reviewed 49 patients who were treated at our institution for pathologic fractures of the subtrochanteric and diaphyseal femurs for group differences in terms of blood loss, length of operation, implant survival, and Musculoskeletal Tumor Society (MSTS) score. RESULTS: We included 49 stabilization procedures of the lower extremity due to pathologic fractures of the proximal or diaphyseal femur, with a mean follow-up of 17.7 months. IM (n = 29) had a significantly shorter operation time than PCO (n = 20) (112.4 ± 9.4 and 163.3 ± 15.96 min, respectively). We did not detect any significant differences in terms of blood loss, complication rate, implant survival, or MSTS score. CONCLUSION: Based on our data, pathologic subtrochanteric and diaphyseal fractures of the femur can be stabilized with IM, which has a shorter operation time than PCO, but the complication rate, implant survival, and blood loss remain unaffected.

Reduced angiogenesis and delayed endochondral ossification in CD163-/- mice highlights a role of M2 macrophages during bone fracture repair.

While recent studies showed that macrophages are critical for bone fracture healing, and lack of M2 macrophages have been implicated in models of delayed union, functional roles for specific M2 receptors have yet to be defined. Moreover, the M2 scavenger receptor CD163 has been identified as a target to inhibit sepsis following implant-associated osteomyelitis, but potential adverse effects on bone healing during blockage therapy have yet to be explored. Thus, we investigated fracture healing in C57BL/6 versus CD163-/- mice using a well-established closed, stabilized, mid-diaphyseal femur fracture model. While gross fracture healing in CD163-/- mice was similar to that of C57BL/6, plain radiographs revealed persistent fracture gaps in the mutant mice on Day 14, which resolved by Day 21. Consistently, 3D vascular micro-CT demonstrated delayed union on Day 21, with reduced bone volume (74%, 61%, and 49%) and vasculature (40%, 40%, and 18%) compared to C57BL/6 on Days 10, 14, and 21 postfracture, respectively (p < 0.01). Histology confirmed large amounts of persistent cartilage in CD163-/- versus C57BL/6 fracture callus on Days 7 and 10 that resolves over time, and immunohistochemistry demonstrated deficiencies in CD206+ M2 macrophages. Torsion testing of the fractures confirmed the delayed early union in CD163-/- femurs, which display decreased yield torque on Day 21, and a decreased rigidity with a commensurate increase in rotation at yield on Day 28 (p < 0.01). Collectively, these results demonstrate that CD163 is required for normal angiogenesis, callus formation, and bone remodeling during fracture healing, and raise potential concerns about CD163 blockade therapy.

Modification to Mirels scoring system location component improves fracture prediction for metastatic disease of the proximal femur.

BACKGROUND: Correctly identifying patients at risk of femoral fracture due to metastatic bone disease remains a clinical challenge. Mirels criteria remains the most widely referenced method with the advantage of being easily calculated but it suffers from poor specificity. The purpose of this study was to develop and evaluate a modified Mirels scoring system through scoring modification of the original Mirels location component within the proximal femur. METHODS: Computational (finite element) experiments were performed to quantify strength reduction in the proximal femur caused by simulated lytic lesions at defined locations. Virtual spherical defects representing lytic lesions were placed at 32 defined locations based on axial (4 axial positions: neck, intertrochanteric, subtrochanteric or diaphyseal) and circumferential (8 circumferential: 45-degree intervals) positions. Finite element meshes were created, material property assignment was based on CT mineral density, and femoral head/greater trochanter loading consistent with stair ascent was applied. The strength of each femur with a simulated lesion divided by the strength of the intact femur was used to calculate the Location-Based Strength Fraction (LBSF). A modified Mirels location score was next defined for each of the 32 lesion locations with an assignment of 1 (LBSF > 75%), 2 (LBSF: 51-75%), and 3 (LBSF: 0-50%). To test the new scoring system, data from 48 patients with metastatic disease to the femur, previously enrolled in a Musculoskeletal Tumor Society (MSTS) cross-sectional study was used. The lesion location was identified for each case based on axial and circumferential location from the CT images and assigned an original (2 or 3) and modified (1,2, or 3) Mirels location score. The total score for each was then calculated. Eight patients had a fracture of the femur and 40 did not over a 4-month follow-up period. Logistic regression and decision curve analysis were used to explore relationships between clinical outcome (Fracture/No Fracture) and the two Mirels scoring methods. RESULTS: The location-based strength fraction (LBSF) was lowest for lesions in the subtrochanteric and diaphyseal regions on the lateral side of the femur; lesions in these regions would be at greatest risk of fracture. Neck lesions located at the anterior and antero-medial positions were at the lowest risk of fracture. When grouped, neck lesions had the highest LBSF (83%), followed by intertrochanteric (72%), with subtrochanteric (50%) and diaphyseal lesions (49%) having the lowest LBSF. There was a significant difference (p < 0.0001) in LBSF between each axial location, except subtrochanteric and diaphyseal which were not different from each other (p = 0.96). The area under the receiver operator characteristic (ROC) curve using logistic regression was greatest for modified Mirels Score using site specific location of the lesion (Modified Mirels-ss, AUC = 0.950), followed by a modified Mirels Score using axial location of lesion (Modified Mirels-ax, AUC = 0.941). Both were an improvement over the original Mirels score (AUC = 0.853). Decision curve analysis was used to quantify the relative risks of identifying patients that would fracture (TP, true positives) and those erroneously predicted to fracture (FP, false positives) for the original and modified Mirels scoring systems. The net benefit of the scoring system weighed the benefits (TP) and harms (FP) on the same scale. At a threshold probability of fracture of 10%, use of the modified Mirels scoring reduced the number of false positives by 17-20% compared to Mirels scoring. CONCLUSIONS: A modified Mirels scoring system, informed by detailed analysis of the influence of lesion location, improved the ability to predict impending pathological fractures of the proximal femur for patients with metastatic bone disease. Decision curve analysis is a useful tool to weigh costs and benefits concerning fracture risk and could be combined with other patient/clinical factors that contribute to clinical decision making.

Callus formation in albino Wistar rats after femur fracture assessed by visible spectroscopy.

Classical histological methods such as hematoxylin-eosin staining, have been, and in some areas still are, an important benchmark for the evaluation of biological tissues. However, the current method of assessment is primarily a qualitative assessment of the tissue under investigation. The aim of this paper is to contribute to the improvement of classical histological methods, by applying physical techniques that allow objective, quantitative data to be added to qualitative assessments, especially in areas where conflicting results are available. To this end, the effect of hypolipidemic medication on the callus formation process of normal bone and pathological osteoporotic bone was investigated. The study allowed us to associate UV-VIS spectroscopy wave number with specific hematoxylin-eosin staining of different types of bone tissue structures, the evolving structures in the callus formation process. This association allowed the quantitative assessment of the callusing process in ovariectomized (associated with pathological, osteoporotic bone) and non-ovariectomized (associated with normal bone) rats, with three groups - the control group, simvastatin-treated group, and fenofibrate-treated group. The study showed that in the non-ovariectomized groups both treatments delayed callus formation. In the ovariectomized groups, simvastatin delayed and fenofibrate promoted callus formation.

The effects of romosozumab combined with active vitamin D3 on fracture healing in ovariectomized rats.

BACKGROUND: In this study, we investigated the potential acceleration of fracture healing and bone mineral density-increasing effects of romosozumab and active vitamin D3 combination therapy for fractures in ovariectomized rats. METHODS: Ovariectomy was performed on 40 24-week-old female Sprague-Dawley rats. After 8 weeks, the rats were subjected to periosteum removal and osteotomy of the femoral shaft followed by osteosynthesis with intramedullary nailing to create fracture models. The rats were then divided into four groups: C group (control), R group (receiving romosozumab at 25 mg/kg once a month via subcutaneous injection), VD group (receiving active vitamin D3 at 0.2 µg/kg twice a week via subcutaneous injection), and R + VD group. Further, 10 rats were included in a sham group. At 10 weeks after the intervention, both femurs were removed and blood samples were collected from all rats. Soft X-ray imaging was used to evaluate bone union, and microcomputed tomography (micro-CT) was used for bone morphometric evaluation. Toluidine blue staining was used for the histopathological evaluation of the undecalcified specimens, and bone turnover marker levels were measured using enzyme-linked immunosorbent assay. RESULTS: Bone morphometry analysis via micro-CT revealed increased mineral density of the trabecular bone in the R + VD group femurs, demonstrating the effectiveness of romosozumab plus active vitamin D3 combination therapy. However, there were no differences in bone union evaluated using soft X-ray imaging, indicating no acceleration of fracture healing. CONCLUSIONS: Although romosozumab and active vitamin D3 combination therapy increased trabecular bone volume, there was no evidence on its ability to accelerate fracture healing.

Risk assessment of femoral pathological fracture in prostate cancer patients by computed tomography analysis.

INTRODUCTION: Prostate cancer often forms osteoblastic lesions that appear as a high-dense shadow upon X-ray. Although the lesions may seem to increase bone strength, pathological fracture occurs in one in four patients with prostate cancer. The aim of this study is to elucidate the factors that may increase the risk of pathological fracture in patients with prostate cancer metastases in the proximal femur by analyzing computed tomography data. MATERIALS AND METHODS: Computed tomography data of the femur of 62 prostate cancer patients were retrospectively analyzed. The patients were divided into three groups based on the presence or absence of femoral metastatic lesions and pathological fracture. Surgical specimens of the proximal femur collected from patients who had a pathological fracture were histologically analyzed. RESULTS: Bone density in the marrow area was increased in all cases with metastases compared with those with no metastases. Contrarily, the cortical bone density at the medial trochanter region was significantly lower in patients who had pathological fractures in the proximal femur than those who did not. Accordingly, histological analysis of the surgical specimens revealed that the affected cortical bone was osteopenic without any apparent new bone formation. CONCLUSION: These results indicate that prostate cancer is less effective in inducing bone formation in the cortex than in the marrow and that the decrease in the cortical bone density at the medial trochanter region leads to an increased risk of pathological fracture. Therefore, a previously undocumented risk factor for pathological fracture in prostate cancer patients is presented.

Outcomes of Intramedullary Nail Fixation for Metastatic Disease: Impending and Pathologic Fractures.

BACKGROUND/AIM: Intramedullary nail (IMN) fixation has become a treatment mean for impending and pathologic femur fractures. Currently there continues to be a lack of data examining functional outcomes, complications, and survivorship of patients treated with IMNs for metastatic disease of the femur. PATIENTS AND METHODS: We retrospectively identified 183 IMNs placed for impending (n=145) or pathologic (n=38) metastatic fractures from 2010 to 2018. Functional outcomes and complications including blood transfusions, venous thromboembolisms (VTEs) and reoperations were studied. RESULTS: Patients with impending lesions were more likely to be ambulatory at final follow-up (pathologic: 82%, impending: 99%, p<0.0001) and reported greater musculoskeletal tumor society scores (p<0.0001). Likewise, pathologic fractures were associated with greater discharge to non-home locations (p<0.0001) and were more likely to require a postoperative transfusion (pathologic: 66%, impending: 22%, p=0.0001). However, there was no difference in the incidence of VTEs (p=1.00) or reoperations (p=0.69) between cohorts. Patients treated for impending fractures had improved overall survival at 1 year (54% vs. 26%, p<0.0001). CONCLUSION: IMN fixation was durable in impending and pathologic femoral fractures. Early identification of metastases remains critical as patients treated for impending lesions had greater functional outcomes, fewer complications and improved survivorship compared to patients treated for pathologic fractures.

Gene Network of Susceptibility to Atypical Femoral Fractures Related to Bisphosphonate Treatment.

Atypical femoral fractures (AFF) are rare fragility fractures in the subtrocantheric or diaphysis femoral region associated with long-term bisphosphonate (BP) treatment. The etiology of AFF is still unclear even though a genetic basis is suggested. We performed whole exome sequencing (WES) analysis of 12 patients receiving BPs for at least 5 years who sustained AFFs and 4 controls, also long-term treated with BPs but without any fracture. After filtration and prioritization of rare variants predicted to be damaging and present in genes shared among at least two patients, a total of 272 variants in 132 genes were identified. Twelve of these genes were known to be involved in bone metabolism and/or AFF, highlighting DAAM2 and LRP5, both involved in the Wnt pathway, as the most representative. Afterwards, we intersected all mutated genes with a list of 34 genes obtained from a previous study of three sisters with BP-related AFF, identifying nine genes. One of these (MEX3D) harbored damaging variants in two AFF patients from the present study and one shared among the three sisters. Gene interaction analysis using the AFFNET web suggested a complex network among bone-related genes as well as with other mutated genes. BinGO biological function analysis highlighted cytoskeleton and cilium organization. In conclusion, several genes and their interactions could provide genetic susceptibility to AFF, that along with BPs treatment and in some cases with glucocorticoids may trigger this so feared complication.

Biomechanical and anatomical considerations for dual plating of distal femur fractures: a systematic literature review.

INTRODUCTION: Distal femur fractures are challenging injuries historically associated with high rates of nonunion and varus collapse with operative management. As a result, clinical and research interest in dual plating (DP) of distal femur fractures has seen a dramatic increase in recent years. The purpose of this study was to systematically review the literature regarding vascular anatomy and biomechanics of distal femur fractures treated with DP constructs. MATERIALS AND METHODS: A systematic literature review of two medical databases (PubMed & Scopus) was performed to identify peer-reviewed studies on the anatomy and biomechanics regarding DP of distal femur fractures. A total of 1,001 papers were evaluated and 14 papers met inclusion criteria (6 anatomy and 8 biomechanics). Methodological quality scores were used to assess quality and potential bias in the included studies. RESULTS: In the biomechanical studies, DP constructs demonstrated greater axial and rotational stiffness, as well as less displacement and fewer incidences of failure compared to all other constructs. Vascular studies showed that the femoral artery crosses the mid-shaft femur approximately 16.0-18.8 cm proximal to the adductor tubercle and it is located on average 16.6-31.1 mm from the femoral shaft at this location, suggesting that medial plate application can be achieved safely in the distal femur. The methodological quality of the included studies was good for biomechanical studies (Traa score 79.1; range 53-92.5) and excellent for anatomical studies (QUACs score 81.9; range 69.0-88.5). CONCLUSIONS: Existing biomechanics literature suggests that DP constructs are mechanically stronger than other constructs commonly used in the treatment of distal femur fractures. Furthermore, medial distal femoral anatomy allows for safe application of DP constructs, even in a minimally invasive fashion. Dual plating should be considered for patients with distal femur fractures that have risk factors for instability, varus collapse, or nonunion.

Differences in femur geometry and bone markers in atypical femur fractures and the general population.

This study aimed to identify differences in femur geometry between patients with subtrochanteric/shaft atypical femur fractures (AFFs) and the general population, and to evaluate the biomechanical factors related to femoral bowing in AFFs. We retrospectively reviewed 46 patients. Data on age, and history and duration of bisphosphonate use were evaluated. Femur computed tomography images were reconstructed into a 3D model, which was analyzed with a geometry analysis program to obtain the femur length, femur width and length, and femoral bowing. Patients were divided into two groups according to fracture location: the subtrochanteric and shaft AFF groups. We compared all parameters between groups, and also between each group and a general population of 300 women ≥ 60 years. Thirty-five patients had a history of bisphosphonate use (average duration, 6.1 years; range, 0.8-20 years). There was no statistical difference in bone turnover markers between the two groups. The shaft AFF group had a lower radius of curvature (ROC) (P = 0.001), lower bone mineral density (BMD, T score) (P = 0.020), and lower calcium (P = 0.016). However, other parameters and rate of bisphosphonate use were not significantly different. There were no significant differences in the parameters of the subtrochanter AFF group and the general population, but the shaft AFF group demonstrated a wider femur width (P < 0.001), longer anteroposterior length (P = 0.001), and lower ROC (P < 0.001) than the general population. Femoral bowing and width increased in shaft AFFs, but similar to subtrochanter AFFs compared to the general population. Our results highlight the biomechanical factors of femur geometry in AFFs.

A Population of M2 Macrophages Associated With Bone Formation.

We previously identified transient brown adipocyte-like cells associated with heterotopic ossification (HO). These ancillary cells support new vessel synthesis essential to bone formation. Recent studies have shown that the M2 macrophage contributes to tissue regeneration in a similar way. To further define the phenotype of these brown adipocyte-like cells they were isolated and characterized by single-cell RNAseq (scRNAseq). Analysis of the transcriptome and the presence of surface markers specific for macrophages suggest that these cells are M2 macrophages. To validate these findings, clodronate liposomes were delivered to the tissues during HO, and the results showed both a significant reduction in these macrophages as well as bone formation. These cells were isolated and shown in culture to polarize towards either M1 or M2 similar to other macrophages. To confirm that these are M2 macrophages, mice received lipopolysacheride (LPS), which induces proinflammation and M1 macrophages. The results showed a significant decrease in this specific population and bone formation, suggesting an essential role for M2 macrophages in the production of bone. To determine if these macrophages are specific to HO, we isolated these cells using fluorescence-activated cell sorting (FACS) from a bone defect model and subjected them to scRNAseq. Surprisingly, the macrophage populations overlapped between the two groups (HO-derived versus callus) suggesting that they may be essential ancillary cells for bone formation in general and not selective to HO. Of further note, their unique metabolism and lipogenic properties suggest the potential for unique cross talk between these cells and the newly forming bone.