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Ischemic osteonecrosis, particularly glucocorticoid-induced osteonecrosis of the femoral head (GIONFH), is primarily due to the dysfunction of osteogenesis and angiogenesis. miRNA, as a therapeutic system with immense potential, plays a vital role in the treatment of various diseases. However, due to the unique microenvironmental structure of bone tissue, especially in the case of GIONFH, where there is a deficiency in the vascular system, it is challenging to effectively target and deliver to the ischemic osteonecrosis area. A drug delivery system assisted by genetically engineered cell membranes holds promise in addressing the challenge of targeted miRNA delivery. Herein, we leverage the potential of miR-21 in modulating osteogenesis and angiogenesis to design an innovative biomimetic nanoplatform system. First, we employed metal-organic frameworks (MOFs) as the core structure to load miR-21-m (miR-21-m@MOF). The nanoparticles were further coated with the membrane of bone marrow mesenchymal stem cells overexpressing CXCR4 (CM-miR-21-m@MOF), enhancing their ability to target ischemic bone areas via the CXCR4-SDF1 axis. These biomimetic nanocomposites possess both bone-targeting and ischemia-guiding capabilities, actively targeting GIONFH lesions to release miR-21-m into target cells, thereby silencing PTEN gene and activating the PI3K-AKT signaling pathway to regulate osteogenesis and angiogenesis. This innovative miRNA delivery system provides a promising therapeutic avenue for GIONFH and potentially other related ischemic bone diseases. 1. CXCR4-Engineered Membranes Enhance Targeting for Ischemic Osteonecrosis. 2. miR-21-Based Gene Therapy for Regulating Osteogenesis and Angiogenesis. 3. Expanding the Use of Membrane-Cloaked MOF Nanoparticles. [Display omitted] [ABSTRACT FROM AUTHOR]

Case: We report a patient with permanent tracheostomy in the context of muscular dystrophy presented for pneumonia. On bronchoscopy, a pedicle screw was seen penetrating the trachea. Computed tomography revealed significant anterior erosion of the T1-T3 vertebrae due to chronic pressure injury from the tracheostomy cuff, exposing pedicle screws implanted >10 years earlier. The patient was fitted for a custom low-pressure cuff. No further sequelae developed for 4 months. Conclusion: To the best of our knowledge, this is the first case of exposed spinal fusion instrumentation due to chronic vertebral erosion. Early sagittal imaging and the use of low-pressure cuffs may help prevent this complication. [ABSTRACT FROM AUTHOR]

BACKGROUND: The pathogenesis of Perthes disease is insidious and easily missed at an early stage, and the mechanism of pathogenesis is still not fully understood. Animal models are an important tool for studying the pathogenesis of Perthes disease and for conducting in vivo experiments. OBJECTIVE: To summarize the relevant domestic and foreign literature, classify animals of different species to review the characteristics and application of animal models of Perthes disease, and provide references for the in vivo experimental research of Perthes disease. METHODS: Using “Perthes, Perthes disease, animal model, animal” as the Chinese and English search terms, articles were searched on PubMed, CNKI, Wanfang, and VIP databases. According to the inclusion and exclusion criteria, 61 articles were finally included. RESULTS AND CONCLUSION: (1) The main species for establishing the animal models of Perthes disease are piglets, rats, mice, young rabbits, puppies, and young sheep. Piglets, rats, young rabbits, young dogs, and young sheep are modeled at the proximal femur, while mice are modeled at the distal femur. (2) So far, piglets are the most used animals to model Perthes disease, and femoral neck ligation is the most mature method to model piglets. The epiphyseal growth plate of rats exists for life, and spontaneously hypertensive rats have the characteristics of primary avascular necrosis of the femoral head. (3) Mice have the advantage of being conducive to gene manipulation, and mice have great application prospects in basic research. The size of the young rabbit is moderate, the size of the femoral head is moderate, and it is easy to observe and obtain materials. (4) The puppies are large and convenient for local operation research, but they are difficult to be kept in captivity and expensive. (5) The imaging changes of young sheep model were not similar to Perthes disease. (6) At present, on the whole, the specific animal and method for modeling are selected according to the experimental conditions and objectives of scholars. The femoral neck cerclage method is the most mature model for piglets, and this model is currently the best. [ABSTRACT FROM AUTHOR]

Ischemic osteonecrosis, particularly glucocorticoid-induced osteonecrosis of the femoral head (GIONFH), is primarily due to the dysfunction of osteogenesis and angiogenesis. miRNA, as a therapeutic system with immense potential, plays a vital role in the treatment of various diseases. However, due to the unique microenvironmental structure of bone tissue, especially in the case of GIONFH, where there is a deficiency in the vascular system, it is challenging to effectively target and deliver to the ischemic osteonecrosis area. A drug delivery system assisted by genetically engineered cell membranes holds promise in addressing the challenge of targeted miRNA delivery. Herein, we leverage the potential of miR-21 in modulating osteogenesis and angiogenesis to design an innovative biomimetic nanoplatform system. First, we employed metal-organic frameworks (MOFs) as the core structure to load miR-21-m (miR-21-m@MOF). The nanoparticles were further coated with the membrane of bone marrow mesenchymal stem cells overexpressing CXCR4 (CM-miR-21-m@MOF), enhancing their ability to target ischemic bone areas via the CXCR4-SDF1 axis. These biomimetic nanocomposites possess both bone-targeting and ischemia-guiding capabilities, actively targeting GIONFH lesions to release miR-21-m into target cells, thereby silencing PTEN gene and activating the PI3K-AKT signaling pathway to regulate osteogenesis and angiogenesis. This innovative miRNA delivery system provides a promising therapeutic avenue for GIONFH and potentially other related ischemic bone diseases. STATEMENT OF SIGNIFICANCE., Competing Interests: Declaration competing of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Age at onset is one of the most important predictors of outcome following ischemic osteonecrosis (ON). Currently, there is no well‐established animal model to study the effects of age on the repair process following ischemic ON. The purpose of this study was to further advance a murine model of ischemic ON using four age groups of mice to determine the effects of aging on revascularization and bone repair following ischemic ON. Ischemia was surgically induced in the distal femoral epiphysis of four age groups of skeletally immature and mature mice; juvenile (5 weeks), adolescent (12 weeks), adult (22 weeks), and middle age (52 weeks). Mice were euthanized at 2 days or 4 weeks post‐ischemia surgery to evaluate the extent of ON, revascularization, and bone repair. Terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling staining showed extensive cell death in the epiphysis of all four age groups at 2 days post‐ischemia surgery. At 4 weeks, the juvenile mice followed by the adolescent mice had significantly greater revascularization and repair of the necrotic marrow space, increased osteoblast and osteoclast numbers, and increased bone formation rates compared to the adult and middle‐age mice. Faster revascularization and bone healing were observed in the skeletally immature mice compared to the skeletally mature mice following ischemic ON. The findings resemble the clinical observation of aging on bone repair following ischemic ON. The mouse model may serve as a useful tool to investigate the mechanisms underlying the age‐related impairment of bone repair in adolescent and adult ON and to develop novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Abstract Introduction Avascular necrosis of the femoral capital epiphysis is the most serious complication after traumatic dislocation of the hip in children. This case report discusses the localization and revascularization of the necrotic femoral head following rarely experienced traumatic open anterior hip dislocation in children. Case presentation Our patient was an 11-year-old Japanese boy who had open anterior hip dislocation sustained in a traffic accident. Reduction of the hip joint was performed in an emergency operation, and he was evaluated using serial gadolinium-enhanced magnetic resonance imaging. T1-weighted magnetic resonance images showed two bands with low signal intensity in the femoral capital epiphysis on coronal and oblique axial planes, indicating the existence of avascular osteonecrosis of the femoral head. We observed gadolinium enhancement in the central region of the epiphysis, where the area between the two bands with low signal intensity was located. Serial assessment with enhanced magnetic resonance images during a non-weight-bearing period of 1.5 years after injury showed revascularization starting from the central region and converging toward the peripheral region. Although the patient had leg-length discrepancy due to the early epiphyseal closure, non-weight-bearing treatment for the avascular osteonecrosis of the femoral head achieved a favorable outcome without any hip joint dysfunction, pain, or sign of secondary osteoarthritic change within 4.5 years after injury. Conclusion We confirmed the revascularization process of the necrotic lesion in the femoral capital epiphysis in an 11-year-old boy using serial gadolinium-enhanced magnetic resonance imaging. Conservative non-weight-bearing treatment achieved a favorable outcome.

ABSTRACT Legg‐Calvé‐Perthes disease (LCPD) is a childhood ischemic osteonecrosis (ON) of the femoral head associated with the elevation of proinflammatory cytokine interleukin‐6 (IL‐6) in the synovial fluid. Currently, there is no effective medical therapy for patients with LCPD. In animal models of ischemic ON, articular chondrocytes produce IL‐6 in response to ischemic ON induction and IL‐6 receptor blockade improves bone healing. High‐mobility group box 1 (HMGB1) is a damage‐associated molecular pattern released from dying cells. In addition, extracellular HMGB1 protein is a well‐known proinflammatory cytokine elevated in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. The purpose of this study was to investigate IL‐6–related proinflammatory cytokines, including HMGB1, in the synovial fluid of patients with LCPD. Our working hypothesis was that HMGB1, produced by articular chondrocytes following ischemic ON, plays an important role in IL‐6 upregulation. Here, HMGB1 protein levels were significantly higher in the synovial fluid of patients with LCPD by threefold compared with controls (p

Ischemic osteonecrosis (ION) can produce permanent deformity and osteoarthritis in the femoral head and other joints. No biologic treatment has been established, and the molecular mechanisms involved in the pathogenesis of ION have not been elucidated. In this work, we found that treatment with sirtuin6 (Sirt6) suppressed inflammatory cytokines, bone resorption, progression of osteoarthritis, and reduced bone deformity in an ION mouse model. We used a deacetylase mutant adenovirus to confirm that those effects were caused by the deacetylase function of Sirt6. Among the osteoclastogenic factors of osteoblasts, only the receptor activator of NF‐κb ligand (RANKL) level changed in response to Sirt6 knockout in primary osteoblasts. In particular, the vitamin D receptor physically interacted with Sirt6 and induced recruitment of Sirt6 around RANKL promoters. Finally, Tg mice overexpressing Sirt6 resisted osteocyte death, bone resorption, and progression of osteoarthritis after ischemic surgery, whereas osteoblast/osteocyte‐specific Sirt6 knockout mice showed aggravated bone loss and severe deformity. Our findings demonstrate that administration of Sirt6 prevents bone loss and osteoarthritis in ischemic conditions. Activation of Sirt6 in osteoblasts/osteocytes could be a new therapeutic approach to treating ION of the femoral head and other bone regions. © 2020 American Society for Bone and Mineral Research (ASBMR). [ABSTRACT FROM AUTHOR]

Legg‐Calvé‐Perthes disease (LCPD) is a childhood ischemic osteonecrosis (ON) of the femoral head associated with the elevation of proinflammatory cytokine interleukin‐6 (IL‐6) in the synovial fluid. Currently, there is no effective medical therapy for patients with LCPD. In animal models of ischemic ON, articular chondrocytes produce IL‐6 in response to ischemic ON induction and IL‐6 receptor blockade improves bone healing. High‐mobility group box 1 (HMGB1) is a damage‐associated molecular pattern released from dying cells. In addition, extracellular HMGB1 protein is a well‐known proinflammatory cytokine elevated in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. The purpose of this study was to investigate IL‐6–related proinflammatory cytokines, including HMGB1, in the synovial fluid of patients with LCPD. Our working hypothesis was that HMGB1, produced by articular chondrocytes following ischemic ON, plays an important role in IL‐6 upregulation. Here, HMGB1 protein levels were significantly higher in the synovial fluid of patients with LCPD by threefold compared with controls (p < 0.05), and were highly correlated with IL‐6 levels (Pearson correlation coefficient 0.94, p < 0.001, R2 = 0.87). In the mouse model of ischemic ON, both HMGB1 gene expression and protein levels were elevated in the articular cartilage. In vitro studies revealed a significant elevation of HMGB1 and IL‐6 proteins in the supernatants of human chondrocytes exposed to hypoxic and oxidative stresses. Overexpressed HMGB1 protein in the supernatants of chondrocytes synergistically increased IL‐6 protein. Silencing HMGB1 RNA in human chondrocytes significantly repressed inteleukin‐1β (IL‐1β) gene expression, but not IL‐6. Further, both IL‐1β and tumor necrosis factor‐α (TNF‐α) protein levels in the synovial fluid of patients with LCPD were significantly correlated with IL‐6 protein levels. Taken together, these results suggest that proinflammatory cytokines, HMGB1, tumor necrosis factor‐α (TNF‐α), and IL‐1β, are significantly involved with IL‐6 in the pathogenesis of LCPD. This study is clinically relevant because the availability of multiple therapeutic targets may improve the development of therapeutic strategy for LCPD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Abstract Legg-Calvé-Perthes disease (LCPD) is a childhood form of ischemic osteonecrosis of the femoral head which can produce a permanent femoral head deformity and early osteoarthritis. The femoral head deformity results from increased bone resorption and decreased bone formation during repair and remodeling of the necrotic femoral head. A recent study showed that a pro-inflammatory cytokine, interleukin-6 (IL-6), is significantly elevated in the synovial fluid of patients with LCPD. We hypothesized that IL-6 elevation decreases bone formation during the repair process following ischemic osteonecrosis and that IL-6 depletion will increase new bone formation. To test this hypothesis, we surgically induced ischemic osteonecrosis in the wild-type (n = 29) and IL-6 knockout (KO) mice (n = 25). The animals were assessed at 48 h, 2 weeks and 4 weeks following the induction of ischemic osteonecrosis using histologic, histomorphometric and micro-CT methods. IL-6 immunohistochemistry showed high expression of IL-6 in the osteonecrotic side of the wild-type mice at 48 h and 4 weeks following ischemic osteonecrosis, but not in the IL-6 KO mice. We also confirmed an undetectable level of IL-6 expression in the primary osteoblasts of the IL-6 KO mice compared to the readily detectable level in the wild-type mice. Furthermore, we confirmed that IL-6 deletion did not affect the extent of bone necrosis in the IL-6 KO mice compared to the wild-type mice by performing histologic and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assessments at 2 weeks following the induction of ischemia. Both groups had the same extent of ischemic osteonecrosis and absence of repair at 2 weeks. At 4 weeks, the necrotic epiphyses showed a significant increase in the extent of revascularization in the IL-6 KO mice compared to the wild-type mice (p = 0.001). In addition, a significantly greater recovery of the hematopoietic bone marrow was observed in the osteonecrotic side of the IL-6 KO mice compared to the wild-type mice (p < 0.01). Vascular endothelial growth factor (VEGF) immunohistochemistry showed regionally increased staining in the areas of repair in the osteonecrosis side of IL-6 KO mice compared to the wild-type mice at 4 weeks following ischemic osteonecrosis. Micro-CT assessment of the wild-type mice at 4 weeks showed a significant decrease in the percent bone volume (p < 0.01) in the osteonecrotic side compared to the control side. In contrast, IL-6 KO mice showed significantly increased bone volume in the osteonecrotic side compared to the osteonecrotic side of WT mice (p < 0.001). No significant difference in the bone volume percentage was found between the control side of the wild-type and the IL-6 KO mice. Histomorphometric analysis at 4 weeks revealed increased osteoblast number/bone surface (p < 0.001), bone formation rate (BFR) (p = 0.0001), and mineral apposition rate (MAR) (p < 0.0001) in the osteonecrotic side of the IL-6 KO mice compared to the wild-type mice. The number of osteoclast/bone surface was also increased in the IL-6 KO mice compared to the wild-type mice (p < 0.0001). No significant difference was observed between the control side of the wild-type and IL-6 KO mice with regards to the number of osteoblast or osteoclast/bone surface, BFR, and MAR. We next obtained primary osteoblasts from IL-6 KO mice and showed they expressed a significantly higher level of RANKL/OPG than wild-type mice (p = 0.001) in hypoxia culture condition. Taken together, the findings indicate that IL-6 deletion stimulates revascularization and new bone formation following ischemic osteonecrosis. This study provides new evidence that therapeutic strategies to block IL-6 may be beneficial for bone healing following ischemic osteonecrosis. Highlights • IL-6 deletion increases revascularization with the recovery of the necrotic marrow space with hematopoietic marrow. • IL-6 knock-out mice show higher bone volume after ischemic osteonecrosis. • IL-6 deletion increases the number of osteoblasts and new bone formation following ischemic osteonecrosis. [ABSTRACT FROM AUTHOR]

ABSTRACT Ischemic osteonecrosis (IO) is caused by disruption of the blood supply to bone. It is a debilitating condition with pathological healing characterized by excessive bone resorption and delayed osteogenesis. Although the majority of research has focused on the role of osteoblasts and osteoclasts in the disease progression, we hypothesize that innate immune cells, macrophages and neutrophils, play a significant role. With the recent development of real-time imaging probes for neutrophils and macrophages, the purpose of this study was to investigate the kinetic immune cell response in a mouse model of IO. Our results show that induction of IO leads to a significant accumulation of activated neutrophils and macrophages at the affected tissue by 48 h after surgery. Additionally, the accumulation of these immune cells remained elevated in comparison to sham controls for up to 6 weeks, indicative of chronic inflammation. Immunohistochemistry confirmed the immune cell infiltration into the necrotic bone marrow and the increased presence of TNFα-positive cells, demonstrating, for the first time, a direct response of these cells to ischemia induced necrotic bone. These new findings support a hypothesis that IO is an osteoimmunologic condition where innate immune cells play a significant role in the chronic inflammation. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:307-313, 2016. [ABSTRACT FROM AUTHOR]

Ischemic osteonecrosis of the femoral head (IOFH) can lead to excessive resorption of the trabecular bone and collapse of the femoral head as a structure. A well-known mineral component to trabecular bone is hydroxyapatite, which can be present in many forms due to ionic substitution, thus altering chemical composition. Unfortunately, very little is known about the chemical changes to bone apatite following IOFH. We hypothesized that the apatite composition changes in necrotic bone possibly contribute to increased osteoclast resorption and structural collapse of the femoral head. The purpose of this study was to assess the macroscopic and local phosphate composition of actively resorbed necrotic trabecular bone to isolate differences between areas of increased osteoclast resorption and normal bone formation. A piglet model of IOFH was used. Scanning electron microscopy (SEM), histology, X-ray absorbance near edge structure (XANES), and Raman spectroscopy were performed on femoral heads to characterize normal and necrotic trabecular bone. Backscattered SEM, micro-computed tomography and histology showed deformity and active resorption of necrotic bone compared to normal. XANES and Raman spectroscopy obtained from actively resorbed necrotic bone and normal bone showed increased carbonate-to-phosphate content in the necrotic bone. The changes in the apatite composition due to carbonate substitution may play a role in the increased resorption of necrotic bone due to its increase in solubility. Indeed, a better understanding of the apatite composition of necrotic bone could shed light on osteoclast activity and potentially improve therapeutic treatments that target excessive resorption of bone. [ABSTRACT FROM AUTHOR]

Abstract: Juvenile ischemic osteonecrosis of the femoral head (IOFH) is one of the most serious hip conditions causing the femoral head deformity. Little is known about BMP signaling following ischemic osteonecrosis. In this study, we found acute BMP2 upregulation in the femoral head cartilage 24h after ischemic induction using our immature pig IOFH model. Similarly, in our ischemic osteonecrosis mouse model, BMP2 expression and BMP signaling were enhanced in the articular cartilage surrounding the necrotic bone. BMP2 was increased in cartilage explants and primary chondrocytes under hypoxia (1% O2) compared with normoxia (21% O2). Addition of the hypoxia inducible factor 1 (HIF1) activator DFO significantly increased BMP2 while HIF1 silencing (siHIF1) only partially reduced BMP2, suggesting other mechanisms of BMP2 upregulation being present. Hypoxia is known to induce the production of free oxygen radicals, which are converted to hydrogen peroxide (H2O2) by superoxide dismutase 2 (SOD2). As an alternative mechanism, we investigated the effect of H2O2/SOD2 production on BMP2 upregulation. Chondrocytes produced more H2O2 under hypoxia than normoxia. H2O2 addition to the chondrocyte culture also significantly increased BMP2 expression. SOD2 was also dramatically increased in the ischemic pig cartilage at 24h following surgery and in primary chondrocytes/cartilage explants culture under hypoxia. SOD2 protein addition to the chondrocyte culture significantly increased BMP2. Moreover, DFO significantly increased SOD2 while HIF1 silencing only partially reduced SOD2. These results suggest that the acute BMP2 response of chondrocytes to ischemic osteonecrosis is more dominantly through the H2O2 production and only partly through the HIF1 pathway. [Copyright &y& Elsevier]

Abstract: Development of a subchondral fracture is one of the earliest signs of structural failure of the immature femoral head following ischemic osteonecrosis, and this eventually leads to a flattening deformity of the femoral head. The mechanical and mineralization changes in the femoral head preceding subchondral fracture have not been elucidated. We hypothesized that ischemic osteonecrosis leads to early material and mechanical alterations in the bone of the subchondral region. The purpose of this investigation was to assess the bone of the subchondral region for changes in the histology of bone cells, microcrack density, mineral content, and nanoindentation properties at an early stage of ischemic osteonecrosis in a piglet model. This large animal model has been shown to develop a subchondral fracture and femoral head deformity resembling juvenile femoral head osteonecrosis. The unoperated, left femoral head of each piglet (n=8) was used as a normal control, while the right side had a surgical ischemia induced by disrupting the femoral neck vessels with a ligature. Hematoxylin and eosin (H&E) staining and TUNEL assay were performed on femoral heads from 3 piglets. Quantitative backscattered electron imaging, nanoindentation, and microcrack assessments were performed on the subchondral region of both control and ischemic femoral heads from 5 piglets. H&E staining and TUNEL assay showed extensive cell death and an absence of osteoblasts in the ischemic side compared to the normal control. Microcrack density in the ischemic side (3.2±0.79 cracks/mm2) was significantly higher compared to the normal side (0.27±0.27 cracks/mm2) in the subchondral region (p<0.05). The weighted mean of the weight percent distribution of calcium (CaMean) also was significantly higher in the ischemic subchondral region (p<0.05). Furthermore, the nanoindentation modulus within localized areas of subchondral bone was significantly increased in the ischemic side (16.8±2.7GPa) compared to the normal control (13.3±3.2GPa) (p<0.05). Taken together, these results support the hypothesis that the nanoindentation modulus of the subchondral trabecular bone is increased in the early stage of ischemic osteonecrosis of the immature femoral head and makes it more susceptible to microcrack formation. We postulate that continued loading of the hip joint when there is a lack of bone cells to repair the microcracks due to ischemic osteonecrosis leads to microcrack accumulation and subsequent subchondral fracture. [Copyright &y& Elsevier]

Abstract: HIF-1α has been shown to be a central mediator of cellular response to hypoxia. The role it plays after ischemic injury to the immature femoral head is unknown. The purpose of this study was to determine the region of the femoral head affected by hypoxia following ischemic injury to the immature femoral head and to determine the site of HIF-1α activation and revascularization. We hypothesize that the epiphyseal cartilage, rather than the bony epiphysis, is the site of HIF-1α activation following ischemic osteonecrosis and that the epiphyseal cartilage plays an important role in the revascularization process. Materials and methods: Femoral head osteonecrosis was surgically induced in 56 immature pigs. Hypoxyprobe staining, cell viability assay, HIF-1α western blot, RT-qPCR of HIF-1α, VEGF, VEGFR2, and PECAM, and micro-CT assessments of microfil-infused femoral heads were performed. Results: Severe hypoxia was present in the bony epiphysis and the lower part of the epiphyseal cartilage following ischemia. In the bony epiphysis, extensive cell death and tissue necrosis was observed with degradation of proteins and RNAs which precluded further analysis. In the epiphyseal cartilage, the loss of cell viability was limited to its deep layer with the remainder of the cartilage remaining viable. Furthermore, the cartilage from the ischemic side showed a significant increase in HIF-1α protein level and HIF-1α expression. VEGF expression in the cartilage was dramatically and significantly increased at 24 h, 2 and 4 weeks (p <0.05 for all) with 5 to 10 fold increase being observed on the ischemic side compared to the normal side. PECAM and VEGFR2 expressions in the cartilage were both significantly decreased at 24 h but returned to the normal levels by 2 and 4 weeks, respectively. Micro-CT showed revascularization of the cartilage on the ischemic side with the vessel volume/total volume equaling the normal side by 4 weeks. Conclusions: Acute ischemic injury to the immature femoral head induced severe hypoxia and cell death in the bony epiphysis and the deep layer of the epiphyseal cartilage. Viable chondrocytes in the superficial layer of the epiphyseal cartilage showed HIF-1α activation and VEGF upregulation with subsequent revascularization occurring in the cartilage. [Copyright &y& Elsevier]

Legg-Calvé-Perthes disease (LCPD) is a childhood ischemic osteonecrosis (ON) of the femoral head associated with the elevation of proinflammatory cytokine interleukin-6 (IL-6) in the synovial fluid. Currently, there is no effective medical therapy for patients with LCPD. In animal models of ischemic ON, articular chondrocytes produce IL-6 in response to ischemic ON induction and IL-6 receptor blockade improves bone healing. High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern released from dying cells. In addition, extracellular HMGB1 protein is a well-known proinflammatory cytokine elevated in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. The purpose of this study was to investigate IL-6-related proinflammatory cytokines, including HMGB1, in the synovial fluid of patients with LCPD. Our working hypothesis was that HMGB1, produced by articular chondrocytes following ischemic ON, plays an important role in IL-6 upregulation. Here, HMGB1 protein levels were significantly higher in the synovial fluid of patients with LCPD by threefold compared with controls ( p  < 0.05), and were highly correlated with IL-6 levels (Pearson correlation coefficient 0.94, p  < 0.001, R 2 = 0.87). In the mouse model of ischemic ON, both HMGB1 gene expression and protein levels were elevated in the articular cartilage. In vitro studies revealed a significant elevation of HMGB1 and IL-6 proteins in the supernatants of human chondrocytes exposed to hypoxic and oxidative stresses. Overexpressed HMGB1 protein in the supernatants of chondrocytes synergistically increased IL-6 protein. Silencing HMGB1 RNA in human chondrocytes significantly repressed inteleukin-1β (IL-1β) gene expression, but not IL-6. Further, both IL-1β and tumor necrosis factor-α (TNF-α) protein levels in the synovial fluid of patients with LCPD were significantly correlated with IL-6 protein levels. Taken together, these results suggest that proinflammatory cytokines, HMGB1, tumor necrosis factor-α (TNF-α), and IL-1β, are significantly involved with IL-6 in the pathogenesis of LCPD. This study is clinically relevant because the availability of multiple therapeutic targets may improve the development of therapeutic strategy for LCPD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., (© 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

Introduction: Legg-Calvé-Perthes disease is a juvenile ischemic osteonecrosis which produces extensive necrotic cell debris and release of damage associated molecular patterns (DAMPs) in the femoral head. The necrotic bone environment induces a chronic inflammatory repair response with excessive bone resorption leading to deformity and early osteoarthritis. Currently there is no minimally invasive method to clear the necrotic materials from the bone to decrease the inflammatory burden of the necrotic environment and to improve the healing process., Hypothesis: We hypothesized that a novel minimally invasive two-needle saline washing technique would be effective to remove cell debris, proteins, and fat from the marrow space of porcine cadaveric humeral heads (HHs)., Materials and Methods: Twenty-two HHs were subjected to three freeze-thaw cycles to simulate osteonecrosis prior to the wash procedure which consisted of placement of two 15-gauge intraosseous needles followed by incremental saline wash. After the washout procedure, the solutions were collected for measurements of turbidity, protein concentration, and cell count. The HHs were analyzed by optical scanning and histology., Results: The solution collected after each wash showed a significant decrease in the turbidity, cell count, and protein concentration (p<0.05). Histologic assessment showed significantly decreased cell debris and adipocytes in the washed group compared to the unwashed group (p<0.001)., Discussion/conclusion: The two-needle intraosseous wash technique effectively removed cell debris and proteins from the marrow space. The technique may be used to reduce the necrotic cell debris and DAMPs present in the necrotic bone., Level of Evidence: III, in vitro comparative study., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

In this study, we aimed to identify a candidate drug that can activate endogenous Angiopoietin 1 (Ang1) expression via drug repositioning as a pharmacological treatment for avascular osteonecrosis. After incubation with 821 drugs from the Food and Drug Administration (FDA)-approved drug library, Ang1 expression in U2OS cell culture media was examined by ELISA. Metformin, the first-line medication for treatment of type 2 diabetes, was selected as a candidate for in vitro and in vivo experimental evaluation. Ang1 was induced, and alkaline phosphatase activity was increased by metformin treatment in U2OS and MG63 cells. Wound healing and migration assay showed increased osteoblastic cell mobility by metformin treatment in U2OS and MG63 cells. Metformin upregulated expression of protein markers for osteoblastic differentiation in U2OS and MG63 cells but inhibited osteoclastic differentiation in Raw264.7 cells. Metformin (25 mg/kg) protected against ischemic necrosis in the epiphysis of the rat femoral head by maintaining osteoblast/osteocyte function and vascular density but inhibiting osteoclast activity in the necrotic femoral head. These findings provide novel insight into the specific biomarkers that are targeted and regulated by metformin in osteoblast differentiation and contribute to understanding the effects of these FDA-approved small-molecule drugs as novel therapeutics for ischemic osteonecrosis.

Synovitis of the affected joint is a common in avascular osteonecrosis (AVN). Increased levels of pro-inflammatory cytokine interleukin-6 (IL-6) have been reported in AVN, but the mechanism of this increase remains unclear. Silent information regulator transcript-1 (SIRT1), an NAD-dependent deacetylase, inhibits the release of inflammatory cytokines. Interferon β (IFN-β) has clear anti-inflammatory properties. We sought to investigate the effects of IFN-β treatment on AVN and to evaluate the specific signal pathway relating to IL-6 and SIRT1 affected during AVN. Using a dissection microscope, AVN was surgically induced in the distal femurs of mice. Exogenous IFN-β was administered to the model mice. The effects of exogenous IFN-β on AVN model mice were assessed using hematoxylin eosin and safranin-O staining, and bone resorption activity was measured using tartrate-resistant acid phosphatase (TRAP) and CD68 staining. Western blots, real-time RT-PCR, and immunohistochemical staining were performed to evaluate the production of SIRT1 and IL-6 in tissues. The RAW 264.7 cell line and bone marrow derived osteoclasts treated with exogenous IFN-β. Histological findings indicated well preserved trabecular bone and decreased osteoclast bone resorption activity in IFN-β treated mice compared with mice in the AVN group. Treatment with IFN-β increased SIRT1 expression and inhibited secretion of IL-6 in this AVN mouse model. IFN-β decreased IL-6 secretion by activating SIRT1 in the RAW 264.7 cell and bone marrow derived osteoclasts. Our work suggests that IFN-β could be used to treat AVN and that both SIRT1 and IL-6 are useful targets for treating patients with AVN., Competing Interests: CONFLICTS OF INTEREST No conflicts of interest to declare.

Background: Legg-Calvé-Perthes Disease (Perthes' disease) is a childhood hip disorder initiated by ischemic necrosis of the growing femoral head. So far, the etiology and pathogenesis of Perthes' disease is poorly understood., Materials and Methods: Avascular osteonecrosis rat model was established to mimic the pathophysiological changes of femoral head necrosis. The chondrocytes of newborn Sprague-Dawley rats were isolated and cultured in hypoxic and normoxic condition. The expression characteristic of the hypoxia-inducible factor-1 alpha (HIF-1α) was evaluated both in vivo and in vitro models. Vascular endothelial growth factor (VEGF) and apoptotic genes in chondrocytes treated with normoxia and hypoxia were also studied., Results: HIF-1α expression increased greatly after ischemic operation and kept at relative high level in the arthromeningitis stage and declined in the stages of osteonecrosis and reconstruction. The HIF-1α mRNA levels of chondrocytes incubated at hypoxia were significantly higher than the cells treated with normoxia at 24 and 72 hours. Hypoxia inhibited VEGF expression; chondrocytes could oppose this inhibition manifested by the increasing of VEGF mRNA level after 72 hours hypoxia. The expression of apoptotic genes, Casp3, Casp8 and Casp9, elevated in chondrocytes after hypoxia with time differences., Conclusion: Hypoxia might be an etiological factor for femoral head necrosis, HIF-1α, VEGF as well as apoptotic genes participated the pathophysiological process of ischemic osteonecrosis.