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.

Differences in bone histomorphometry between White postmenopausal women with and without atypical femoral fracture after long-term bisphosphonate therapy.

Bone histomorphometric endpoints in transilial biopsies may be associated with an increased risk of atypical femoral fracture (AFF) in patients with osteoporosis who take antiresorptives, including bisphosphonates (BPs). One way to test this hypothesis is to evaluate bone histomorphometric endpoints in age-, gender-, and treatment time-matched patients who either had AFF or did not have AFF. In this study, we performed transiliac bone biopsies in 52 White postmenopausal women with (n = 20) and without (n = 32) AFFs, all of whom had been treated for osteoporosis continuously with alendronate for 4-17 yr. Despite the matched range of treatment duration (4-17 yr), AFF patients received alendronate for significantly longer time (10.7 yr) than non-AFF patients (8.0 yr) (P = .014). Bone histomorphometric endpoints reflecting microstructure and turnover were assessed in cancellous, intracortical, and endocortical envelopes from transilial biopsy specimens obtained from BP-treated patients 3-6 mo after AFF and from non-AFF patients with similar age-, gender-, and range of BP treatment duration. However, in both cancellous and intracortical envelopes, AFF patients had significantly lower wall thickness (W.Th) and higher osteoclast surface (Oc.S/BS) than non-AFF patients. In addition, AFF patients had significantly higher eroded surface (ES/BS) only in the intracortical envelope. None of the dynamic variables related to bone formation and turnover differed significantly between the groups. In conclusion, in the ilium of BP-treated patients with osteoporosis, AFF patients have lower thickness of superficial bone (lower W.Th) of the cancellous and cortical envelopes than non-AFF patients. AFF and non-AFF patients have a similar bone turnover rate in the ilium. Furthermore, in this population, as in previous work, AFF is more likely to occur in BP-treated patients with longer treatment duration.

Bisphosphonates (BPs) are widely used to prevent osteoporotic fracture and treat osteoporosis. However, prolonged use of BPs may increase the risk of atypical femoral fracture (AFF), and their pathogenesis remains unclear. This study compared the bone histomorphometric findings in cancellous and cortical bones between White osteoporotic women with (n = 20) and without AFF (n = 32), who had received BP treatment for a matched duration of 4­17 yr. The BP-treated patients with AFF had significantly lower wall thickness (W.Th) in both cancellous and cortical bones compared to BP-treated patients without AFF. There were no significant differences in bone formation, turnover, or mineral apposition rate between BP-treated AFF and non-AFF patients. In conclusion, our study results suggest that AFF risk is increased in BP-treated patients with smaller young and healthy superficial bone areas (indicated by lower W.Th). Surprisingly, we also discovered that patients with and without AFF have similar bone turnover rates, which contradicts previous beliefs. Our findings provide valuable insights into the potential factors contributing to AFF in BP-treated patients.

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.

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.

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.

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.

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.

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.

Free Transplantation of a Tissue Engineered Bone Graft into an Irradiated, Critical-Size Femoral Defect in Rats.

Healing of large bone defects remains a challenge in reconstructive surgery, especially with impaired healing potential due to severe trauma, infection or irradiation. In vivo studies are often performed in healthy animals, which might not accurately reflect the situation in clinical cases. In the present study, we successfully combined a critical-sized femoral defect model with an ionizing radiation protocol in rats. To support bone healing, tissue-engineered constructs were transferred into the defect after ectopic preossification and prevascularization. The combination of SiHA, MSCs and BMP-2 resulted in the significant ectopic formation of bone tissue, which can easily be transferred by means of our custom-made titanium chamber. Implanted osteogenic MSCs survived in vivo for a total of 18 weeks. The use of SiHA alone did not lead to bone formation after ectopic implantation. Analysis of gene expression showed early osteoblast differentiation and a hypoxic and inflammatory environment in implanted constructs. Irradiation led to impaired bone healing, decreased vascularization and lower short-term survival of implanted cells. We conclude that our model is highly valuable for the investigation of bone healing and tissue engineering in pre-damaged tissue and that healing of bone defects can be substantially supported by combining SiHA, MSCs and BMP-2.

Fibrous Demineralized Bone Matrix (DBM) Improves Bone Marrow Mononuclear Cell (BMC)-Supported Bone Healing in Large Femoral Bone Defects in Rats.

Regeneration of large bone defects is a major objective in trauma surgery. Bone marrow mononuclear cell (BMC)-supported bone healing was shown to be efficient after immobilization on a scaffold. We hypothesized that fibrous demineralized bone matrix (DBM) in various forms with BMCs is superior to granular DBM. A total of 65 male SD rats were assigned to five treatment groups: syngenic cancellous bone (SCB), fibrous demineralized bone matrix (f-DBM), fibrous demineralized bone matrix densely packed (f-DBM 120%), DBM granules (GDBM) and DBM granules 5% calcium phosphate (GDBM5%Ca2+). BMCs from donor rats were combined with different scaffolds and placed into 5 mm femoral bone defects. After 8 weeks, bone mineral density (BMD), biomechanical stability and histology were assessed. Similar biomechanical properties of f-DBM and SCB defects were observed. Similar bone and cartilage formation was found in all groups, but a significantly bigger residual defect size was found in GDBM. High bone healing scores were found in f-DBM (25) and SCB (25). The application of DBM in fiber form combined with the application of BMCs shows promising results comparable to the gold standard, syngenic cancellous bone. Denser packing of fibers or higher amount of calcium phosphate has no positive effect.

Effects of different vardenafil doses on bone healing in a rat fracture model.

OBJECTIVES: We aimed to investigate the radiological, biomechanical, histopathological, histomorphometric, and immunohistochemical effects of different doses of vardenafil on fracture healing. MATERIALS AND METHODS: Fifty-one rats were divided into three groups. Group V5 was given 5 mg/kg/day of vardenafil; Group V10 was given 10 mg/kg/day of vardenafil; and the control group was given the same volume of saline. Six rats from each group were sacrificed on Day 14 (early period) and the remaining rats were sacrificed on Day 42 (late period). Callus/femoral volume and bone mineral density were measured using micro-computed tomography. Five femurs from each group in the late period were examined by biomechanical tests. In addition to the histopathological and histomorphometric evaluations, immunohistochemical analyses were performed to examine the levels of inducible nitric oxide synthase (iNOS), transforming growth factor-3 (TGF-ß3), and nuclear factor kappa B (NF-κB) proteins. RESULTS: Both doses of vardenafil increased primary bone volume and maximal bone fracture strength in late period, compared to the control group (p<0.05). Histological healing scores of vardenafil groups were significantly higher in early period (p<0.001). While cartilaginous callus/total callus ratio in early period was higher, callus diameter/femoral diameter ratio in late period was lower in vardenafil groups (p<0.01). The NF-κB immunopositivity in V10 group decreased in early period, compared to control group (p<0.001). The TGF-ß3 and iNOS immunopositivity increased in both V5 and V10 groups, compared to the control group in early period, but returned to normal in late period. CONCLUSION: During the first period of fracture healing process in which vasodilation is mostly required with increasing inflammation, vardenafil has ameliorating effects on the bone union and supports fracture healing.

Is an anodizing coating associated to the photobiomodulation able to optimize bone healing in ovariectomized animal model?

This in vivo study investigated whether the bioactivity of anodizing coating, produced by plasma electrolytic oxidation (PEO), on mini-plate in femur fracture could be improved with the association of photobiomodulation (PBM) therapy. From the 20 ovariectomized Wistar female rats, 8 were used for model characterization, and the remaining 12 were divided into four groups according to the use of PBM therapy by diode laser (808 nm; power: 100 mW; energy: 6.0 J; energy density: 212 J/cm2; power density: 3.5 W/cm2) and the type of mini-plate surface (commercially pure titanium mini-plate -cpTi- and PEO-treated mini-plate) as follow: cpTi; PEO; cpTi/PBM; and PEO/PBM. After 60 days of surgery, fracture healing underwent microstructural, bone turnover, histometric, and histologic adjacent muscle analysis. Animals of groups with PEO and PBM showed greater fracture healing than cpTi control group under histometric and microstructural analysis (P < 0.05); however, bone turnover was just improved in PBM's groups (P < 0.05). there was no difference between cpTi and PEO without PBM (P > 0.05). Adjacent muscle analysis showed no metallic particles or muscle alterations in all groups. PEO and PBM are effective strategies for bone repair in fractures, however their association does not provide additional advantages.

Role of microRNA-335 carried by bone marrow mesenchymal stem cells-derived extracellular vesicles in bone fracture recovery.

Mesenchymal stem cells (MSCs) have the potential to reduce healing time and treat nonunion in fracture patients. In this study, bone marrow MSCs-derived extracellular vesicles (B-EVs) were firstly extracted and identified. CD9-/- and normal mice were enrolled for the establishment of fracture models and then injected with B-EVs. Osteoblast differentiation and fracture recovery were estimated. The levels of osteoblast-related genes were detected, and differentially expressed microRNAs (miRs) in B-EVs-treated normal fracture mice were screened and verified. The downstream mechanisms of miR were predicted and assessed. The loss-of functions of miR-335 in B-EV and gain-of-functions of VapB were performed in animal and cell experiments to evaluate their roles in bone fracture. Collectively, B-EVs promoted bone fracture recovery and osteoblast differentiation by releasing miR-335. miR-335 downregulation in B-EVs impaired B-EV functions in fracture recovery and osteoblast differentiation. miR-335 could target VapB, and VapB overexpression reversed the effects of B-EVs on osteoblast differentiation. B-EV treatment activated the Wnt/ß-catenin pathway in fracture mice and osteoblasts-like cells. Taken together, the study suggested that B-EVs carry miR-335 to promote bone fracture recovery via VapB and the Wnt/ß-catenin pathway. This study may offer insights into bone fracture treatment.

miR-155-5p regulates mesenchymal stem cell osteogenesis and proliferation by targeting GSK3B in steroid-associated osteonecrosis.

microRNAs (miRNAs) have recently been recognized as playing an important role in bone-associated diseases. This study investigated whether the reduced miR-155-5p in steroid-associated osteonecrosis of the femoral head (ONFH) attenuated osteogenic differentiation and cell proliferation by targeting GSK3B. Bone marrow was collected from the proximal femurs of patients with steroid-associated ONFH (n = 10) and patients with new femoral neck fracture (n = 10) and mesenchymal stem cells (MSCs) were isolated. The expression profile, the biological function of miR-155-5p, and the interaction between miR-155-5p and GSK3B were investigated by cell viability measurement, western blot, real-time polymerase chain reaction, luciferase reporter assay, and Alizarin Red S (ARS) staining of MSCs. The MSCs that were obtained from the femoral neck fracture group and from the steroid-associated ONFH group were transfected with or without miR-155-5p. We found that, in ONFH samples, the level of mature miR-155-5p was significantly lower than that of control samples. By inhibiting GSK3B, miR-155-5p promoted the nuclear translocation of ß-catenin, increased the expression of osteogenesis-related genes, and facilitated the proliferation and differentiation of MSCs. Restoring the expression of GSK3B in MSCs partially reversed the effect of miR-155-5p. These findings suggest that reduced miR-155-5p in steroid-associated ONFH attenuates osteogenic differentiation and cell proliferation by increased levels of GSK3B and inhibition of Wnt signaling.

Protective Effect of Curcumin on Bone Trauma in a Rat Model via Expansion of Myeloid Derived Suppressor Cells.

BACKGROUND Bone fracture, a common injury to bones leads to various biophysiological changes and pathological responses in the body. The current study investigated curcumin for treatment of bone fracture in a rat model of bone trauma, and evaluated the related mechanism. MATERIAL AND METHODS The rats were separated randomly into 3 groups; sham, model, and curcumin treatment groups. The fracture rat model was established by transverse osteotomy in the right femur bone at the mid-shaft. The osteoblast count was determined using hematoxylin and eosin staining. Vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) expression were measured by western blotting. RESULTS The rpS6-phosphorylation was suppressed and light chain 3 (LC3II) expression elevated in the curcumin treated group of the fracture rat model. In the curcumin-treated group, mineralization of fracture calluses was markedly higher on day 14 of fracture. The formation of osteoblasts was observed at a greater rate in the curcumin treated group compared to the model rat group. Treatment of rats with curcumin significantly (P<0.05) promoted expression of PCNA and VEGF. The decrease in CD11b+/Gr-1+ cell expansion in rats with bone trauma was alleviated significantly by curcumin treatment. A marked increase in arginase-1 expression in rats with bone trauma was caused by curcumin treatment. CONCLUSIONS In summary, curcumin activates autophagy and inhibits mTOR activation in bone tissues of rats with trauma. The curcumin promoted myeloid-derived suppressor cell (MDSC) proliferation and increased expansion of MDSCs in a rat model of trauma. Therefore, curcumin may have beneficial effect in patients with bone trauma and should be evaluated further for development of treatment.