Ischemic Osteonecrosis Research Articles

Chemotactic recruitment of genetically engineered cell membrane-camouflaged metal−organic framework nanoparticles for ischemic osteonecrosis treatment

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 significance1.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.

Cellular senescence is associated with osteonecrosis of the femoral head while mesenchymal stem cell conditioned medium inhibits bone collapse

Osteonecrosis of the femoral head (ONFH) is a type of ischemic osteonecrosis that causes pain, loss of function, and femoral head collapse. Here, we analyzed samples of femoral heads excised from patients with ONFH to clarify the relationship between ischemic osteonecrosis and cellular senescence. X-gal staining was strong and p16INK4a-positive cells were abundant in the transitional region of ONFH. The β-galactosidase-positive cells in the transitional region were also positive for nestin, periostin, or DMP-1. In contrast, no β-galactosidase-positive cells were detected in the healthy region. The senescence-associated p16INK4a, p21, and p53 were upregulated in ONFH tissue. We also examined and analyzed a mouse ischemic femoral osteonecrosis model in vivo to verify the association between ONFH and cellular senescence. Human mesenchymal stem cell-conditioned medium (MSC-CM) was administered to determine its therapeutic efficacy against cellular senescence and bone collapse. MSC-CM reduced the number of senescent cells and downregulated the aforementioned senescence-related genes. It also decreased the number of empty lacunae 4 weeks after ischemia induction and promoted bone formation. At 6 weeks post-surgery, MSC-CM increased the trabecular bone volume, thereby suppressing bone collapse. We conclude that cellular senescence is associated with ONFH and that MSC-CM suppresses bone collapse in this disorder.

Anti-sclerostin antibody therapy prevents post-ischemic osteonecrosis bone collapse via interleukin-6 association

Osteonecrosis of the femoral head (ONFH) is a debilitating condition characterized by subchondral bone necrosis, which frequently culminates in joint destruction. Although total hip arthroplasty is conventionally practiced to remediate ONFH, for patients under the age of 60, the outcomes can be suboptimal. Chronic inflammation, particularly that mediated by interleukin-6 (IL-6), has been conjectured to be a potential mechanism underlying the etiology of ONFH. This study aimed at exploring the interplay between IL-6, the canonical Wnt signaling pathway, and ONFH to provide insights for potential therapeutic interventions. Human ONFH specimens depicted an elevation in β-catenin expression in the transitional layer, while IL-6 levels were pronounced in the same region. Subsequently, mouse models of ischemic osteonecrosis were treated with an anti-sclerostin antibody to assess its effects on bone metabolism and cellular processes. Histological analysis revealed that the administration of anti-sclerostin antibodies effectuated early recovery from bone necrosis, reduced empty lacunae, and suppressed IL-6 expression. The treatment evidently initiated the activation of the Wnt/β-catenin signaling pathway, presenting a potential mechanism associated with IL-6-mediated inflammation. Furthermore, the antibody upregulated osteoblast formation, downregulated osteoclast formation, and increased bone volume. Micro-CT imaging demonstrated increased bone volume, prevented epiphyseal deformity, and improved compression strength.Therefore, this study yields significant findings, indicating the potency of anti-sclerostin antibodies in effectively modulating the Wnt/β-catenin pathway, associating with IL-6 expression, and preventing post-ONFH bone collapse. Additionally, this preclinical investigation in mouse models offers an avenue for prospective research on potential therapeutic interventions against human ONFH.

Risk factors for ischemic osteonecrosis of the femoral head after internal fixation with multiple cannulated screws for Pauwels type III femoral neck fracture

Background/Aim. Numerous factors lead to hip fractures that are common and often devastating in the geriatric population. The aim of the study was to determine the risk factors for ischemic osteonecrosis of the femoral head (ONFH) in patients after internal fixation with multiple cannulated screws for Pauwels type III femoral neck fracture (FNF). Methods. A total of 212 patients with Pauwels type III FNF, who underwent internal fixation with multiple cannulated screws from January 2019 to September 2022 and received one-year follow-up after surgery, were selected. Based on their clinical data and the incidence of ONFH, they were divided into two groups: the group with ischemic ONFH occurrence (n = 30) and the ischemic ONFH non-occurrence group (n = 182). Logistic regression analysis was conducted to analyze the risk factors for postoperative ischemic ONFH. Results. Age, body mass index (BMI), time from injury to surgery, partial weight bearing (PWB) time after surgery, and preoperative diabetes mellitus (DM) were significantly different between the ischemic ONFH occurrence and ischemic ONFH non-occurrence groups (p < 0.05), while other clinical data showed no significant differences (p > 0.05). The results of logistic regression analysis revealed that age (> 55 years), PWB time after surgery (? 3 months), BMI (? 24 kg/m2), time from injury to surgery (> 2 days), and preoperative DM were independent risk factors for postoperative ischemic ONFH (p < 0.05). Conclusion. The incidence of postoperative ischemic ONFH can be prevented by effectively controlling the following factors: age, BMI, PWB after surgery, and preoperative DM.

Interleukin-6 receptor blockade improves bone healing following ischemic osteonecrosis in adolescent mice

ObjectiveJuvenile ischemic osteonecrosis (JIO) of the femoral head is one of the most serious hip disorders causing a permanent deformity of the femoral head in childhood. We recently reported that interleukin 6 (IL-6) is significantly increased in the hip synovial fluid of patients with JIO and that articular chondrocytes are primary source of IL-6. Adolescent JIO is particularly challenging to treat and has poor outcome. This study determined if IL-6 receptor blockade prevents bone loss and improves the bone healing in adolescent JIO. MethodAdolescent mice (12-week-old) surgically induced with JIO were treated with either saline or MR16-1, an IL-6 receptor blocker. ResultsMicro-CT assessment showed significantly increased bone volume (p ​< ​0.001, Cohen's d ​= ​2.0) and trabecular bone thickness (p ​< ​0.001, d ​= ​2.3) after the MR16-1 treatment. Histomorphometric assessment showed significantly increased osteoblast number (p ​< ​0.01, d ​= ​2.3), bone formation rate (p ​< ​0.01, d ​= ​4.3), and mineral apposition rate (p ​< ​0.01, d ​= ​4.1) after the MR16-1 treatment. The number of osteoclasts was unchanged. Histologic assessment showed significantly increased revascularization (p ​< ​0.01) and restoration of the necrotic marrow with new hematopoietic bone marrow (p ​< ​0.01). Vascular endothelial growth factor (VEGF) expression was increased in the revascularized area and the articular cartilage, and in the cultured chondrocytes treated with IL-6 receptor inhibitor. ConclusionIL-6 blockade in adolescent mice with JIO enhanced bone formation and revascularization. The findings suggest IL-6 receptor blocker as a potential medical therapy for adolescent JIO.

Gain-of-Function of FGFR3 Accelerates Bone Repair Following Ischemic Osteonecrosis in Juvenile Mice.

Bone collapse, bone deformity, and a long treatment period are major clinical problems associated with juvenile ischemic osteonecrosis (JIO). Accelerating the process of bone repair in JIO is expected to shorten the treatment duration and better maintain morphology. We previously indicated that both bone formation and resorption were accelerated following distraction osteogenesis-mediated limb lengthening in genetically engineered mutant mice with a gain-of-function mutation in fibroblast growth factor receptor 3 (FGFR3) gene (i.e., Fgfr3 mice). The purpose of this study was to investigate the role of FGFR3 in the bone repair process following surgically induced ischemic osteonecrosis in the mutant mice. Epiphyseal deformity was less in the Fgfr3 mice compared to the wild-type mice at 6weeks following ischemic osteonecrosis in skeletally immature age. Assessment of the morphology by micro-computed tomography (CT) revealed that the trabecular bone volume was increased in the Fgfr3 mice. Dynamic bone histomorphometry revealed increased rates of bone formation and mineral apposition in the Fgfr3 mice at 4weeks post-surgery. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells rapidly increased, and the numbers of TdT-mediated dUTP nick-end labeling (TUNEL)-positive cells rapidly decreased in the Fgfr3 mice. Vascular endothelial growth factor (VEGF) expression was increased at the earlier phase post-surgery in the Fgfr3 mice. The activation of FGFR3 signaling shortens the time needed for bone repair after ischemic osteonecrosis by accelerating revascularization, bone resorption, and new bone formation. Our findings are clinically relevant as a new potential strategy for the treatment of JIO.

A novel model of traumatic femoral head necrosis in rats developed by microsurgical technique

BackgroundClinical angiography and vascular microperfusion confirmed that the femoral head retains blood supply after a collum femur fracture. However, no animal model accurately mimics this clinical situation. This study was performed to establish a rat model with retained viability of the femoral head and partial vasculature deprivation-induced traumatic caput femoris necrosis by surgery.MethodsThirty rats were randomly divided into three groups (n = 10 per group): normal group, sham-operated group (Control), and ischemic osteonecrosis group. The femoral head of the normal group of rats underwent a gross anatomy study and microangiography to identify femoral head blood supply. Microsurgical techniques were used to cauterize the anterior-superior retinacular vessels to induce osteonecrosis. Hematoxylin and Eosin (H&E) staining were used for femoral head histologic assessment. Morphologic assessments of the deformity in and trabecular bone parameters of the femoral head epiphysis were performed using micro-CT.ResultsThe blood supply of the femoral head in rats primarily came from the anterior-superior, inferior, and posterior retinacular arteries. However, anterior-superior retinacular vasculature deprivation alone was sufficient in inducing femoral head osteonecrosis. H&E showed bone cell loss in nuclear staining, disorganized marrow, and trabecular structure. The bone volume (BV) decreased by 13% and 22% in the ischemic group after 5 and 10 weeks, respectively. The mean trabecular thickness (Tb.Th) decreased from 0.09 to 0.06 mm after 10 weeks. The trabecular spacing (Tb.Sp) increased from 0.03 to 0.05 mm after 5 weeks, and the epiphyseal height-to-diameter (H/D) ratio decreased.ConclusionsWe developed an original and highly selective rat model that embodied femoral head traumatic osteonecrosis induced by surgical anterior-superior retinacular vasculature deprivation.

Damage associated molecular patterns in necrotic femoral head inhibit osteogenesis and promote fibrogenesis of mesenchymal stem cells.

In Legg-Calvé-Perthes disease (LCPD), a loss of blood supply to the juvenile femoral head leads to extensive cell death and release of damage-associated molecular patterns (DAMPs). Over time chronic inflammatory repair process is observed with impaired bone regeneration. Increased fibrous tissue and adipose tissue are seen in the marrow space with decreased osteogenesis in a piglet model of LCPD, suggesting inhibition of osteoblastic differentiation and stimulation of fibroblastic and adipogenic differentiation of mesenchymal stem cell (MSC) during the healing process. Little is known about the DAMPs present in the necrotic femoral head and their effects on MSC differentiation. The purpose of this study was to characterize the DAMPs present in the femoral head following ischemic osteonecrosis and to determine their effects on MSC differentiation. Necrotic femoral heads were flushed with saline at 48h, 2weeks and 4weeks following the induction of ischemic osteonecrosis in piglets to obtain necrotic bone fluid (NBF). Western blot analysis of the NBF revealed the presence of prototypic DAMP, high mobility group box 1 (HMGB1), and other previously described DAMPs: biglycan, 4-hydroxynonenal (4-HNE), and receptor activator of NF-κB ligand (RANKL). ELISA of the NBF revealed increasing levels of inflammatory cytokines IL1β, IL6 and TNFα with the temporal progression of osteonecrosis. To determine the effects of NBF on MSC differentiation, we cultured primary porcine MSCs with NBF obtained by in vivo necrotic bone flushing method. NBF inhibited osteoblastic differentiation of MSCs with significantly decreased OSX expression (p=0.008) and Von Kossa/Alizarin Red staining for mineralization. NBF also significantly increased the expression of proliferation markers Ki67 (p=0.03) and PCNA (p<0.0001), and fibrogenic markers Vimentin (p=0.02) and Fibronectin (p=0.04). Additionally, NBF treated MSC cells showed significantly elevated RANKL/OPG secretion ratio (p=0.003) and increased expression of inflammatory cytokines IL1β (p=0.006) and IL6 (p<0.0001). To specifically assess the role of DAMPs in promoting the fibrogenesis, we treated porcine fibroblasts with artificial NBF produced by bone freeze-thaw method. We found increased fibroblastic cell proliferation in an NBF dose-dependent manner. Lastly, we studied the effect of HMGB1, a prototypic DAMP, and found that HMGB1 partially contributes to MSC proliferation and fibrogenesis. In summary, our findings show that DAMPs and the inflammatory cytokines present in the necrotic femoral head inhibit osteogenesis and promote fibrogenesis of MSCs, potentially contributing to impaired bone regeneration following ischemic osteonecrosis as observed in LCPD.

Development of a murine model of ischemic osteonecrosis to study the effects of aging on bone repair.

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 fourage 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-agemice. 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.

Minimally Invasive Necrotic Bone Washing Improves Bone Healing After Femoral Head Ischemic Osteonecrosis: An Experimental Investigation in Immature Pigs.

Ischemic osteonecrosis of the femoral head produces necrotic cell debris and inflammatory molecules in the marrow space, which elicit a chronic inflammatory repair response. The purpose of this study was to determine the effects of flushing out the necrotic cell debris and inflammatory proteins on bone repair in a piglet model of ischemic osteonecrosis. Osteonecrosis of the femoral head of the right hindlimb was induced in 12 piglets by tying a ligature tightly around the femoral neck. One week after the surgery, 6 animals were treated with a percutaneous 3-needle bone washing procedure and non-weight-bearing (NWB) of the right hindlimb (wash group). The total saline solution wash volume was 450 mL per femoral head. Serial wash solutions were collected and analyzed. The remaining 6 animals were treated with NWB only (NWB group). At 8 weeks after the surgery, the femoral heads were assessed using radiography, micro-computed tomography (micro-CT), and histological analysis. In addition, we compared the results for these piglets with our published results for 6 piglets treated with multiple epiphyseal drilling (MED) plus NWB without bone washing (MED group). Necrotic cells and inflammatory proteins were present in the bone wash solution collected 1 week after ischemia induction. The protein and triglyceride concentrations decreased significantly with subsequent washing (p < 0.005). At 8 weeks after ischemia induction, the wash group had a significantly higher bone volume than the MED or NWB group (p < 0.0001). Histological bone-formation measures were also significantly increased in the wash group compared with the MED group (p = 0.002) or NWB group (p < 0.0001) while macrophage numbers were significantly decreased in the wash group. The percutaneous 3-needle procedure flushed out cell debris and inflammatory proteins from the necrotic femoral heads, decreased osteoclasts and macrophages, and increased bone formation following induction of ischemic osteonecrosis. We believe that this is the first study to investigate the concept of washing out the necrotic femoral head to improve bone healing. The minimally invasive procedure may be useful to improve the necrotic bone environment and bone repair following ischemic osteonecrosis.

A rat model of ischemic osteonecrosis for investigating local therapeutics using biomaterials

Osteonecrosis is one of the most disabling diseases affecting pediatric and adult populations. Local application of biomaterials is a promising therapeutic strategy for osteonecrosis. Currently, there is a lack of low-cost animal models of osteonecrosis for testing and developing biomaterials-driven therapeutics. To develop a rat model of ischemic osteonecrosis (IO), the distal femoral epiphysis was selected due to its size 7.7 folds larger than the proximal femoral epiphysis (p<0.0001). The feasibility of intraosseous drillings and the local application of biomaterials were determined. Four model biomaterials were successfully applied: injectable hydrogel, microsphere, bone cement, and implant. The IO was induced by surgically cauterizing the blood vessels supplying the distal femoral epiphysis. Osteonecrosis of the whole epiphysis was achieved with a complete absence of blood flow and near 100% of apoptotic osteocytes. At eight weeks after IO, severe bone deformity and osteoarthritis developed in the affected epiphysis. The histological analysis showed 50% lacunae empty in the IO group compared to 2% in the control group (p<0.0001). The μCT analysis showed the epiphyseal quotient decreased to 0.46 in the IO group compared to 0.53 in the control group (p<0.0001), and the distal femoral epiphysis in the IO group was 19% smaller than the control group (p<0.01). The Safranin O stained sections showed articular cartilage erosions and subchondral bone fractures in the IO group. In summary, we established a clinically relevant IO model on rats that is compatible with the application of biomaterials for treatment. Statement of significanceOsteonecrosis is one of the most serious orthopedic conditions, leading to permanent joint deformity and end-stage osteoarthritis. An efficient and low-cost animal model is essential for development and testing of new treatment strategies for osteonecrosis. This is the first study to develop a clinically relevant model of osteonecrosis on the distal femoral epiphysis of rats. The model is highly efficient in developing osteonecrosis with relatively low cost and it provides suitable skeletal size to apply various forms of biomaterials. More importantly, it mimicked the pathological features and progression of osteonecrosis in humans. The study is expected to have an important impact on the development and testing of innovative biological therapeutics for osteonecrosis.

Причины развития остеоартрита тазобедренного сустава у пациентов с ювенильным идиопатическим артритом: результаты ретроспективного исследования и анализ литературных данных

Osteoarthritis (OsA) of the hip joint (HJ) is one of the most serious structural complications of juvenile idiopathic arthritis (JIA). Objective. Assess risk factors for OsA of the HJ in patients with JIA. Patients and methods. This retrospective study included 753 JIA patients aged 2 to 17 years. The diagnosis of JIA was based on the ILAR criteria. The diagnosis of HJ OsA (n = 48) was based on the Dale radiological classification; the indications for joint replacement were formed according to the WOMAC scale and Oxford hip score. Results. The development of HJ OsA was registered within 5 years (range 2,4; 9,4 years) after JIA onset in 6.4% of patients with JIA (n = 48) or 31.4% of patients who had hip joint lesions. The frequency of HJ replacement (n = 16) was 2.1%, 10.5%, 33.3% in all children with JIA, among patients with HJ arthritis, and patients with HJ osteoarthritis, respectively. The main predictors of HJ OsA included systemic arthritis, administration of systemic corticosteroids, &gt;4 active joints, total alkaline phosphatase &lt;165 U/L, long time without remission, involvement of joints of the upper half of the body, and no timely initiation of biologicals. The predictors of HJ replacement were systemic arthritis, pulse therapy with methylprednisolone, and late administration of biologicals. Conclusion. Timely control of JIA activity, achievement of remission, avoidance of glucocorticosteroid therapy or its early replacement with biologicals reduced the risks of developing structural lesions of the HJ in patients with JIA. Key words: osteoarthritis, hip joint, ischemic osteonecrosis, juvenile idiopathic arthritis, risk factors, corticosteroids

Strontium-calcium phosphate hybrid cement with enhanced osteogenic and angiogenic properties for vascularised bone regeneration.

Vascularized bone tissue engineering is regarded as one of the optimal treatment options for large bone defects. The lack of angiogenic properties and unsatisfactory physicochemical performance restricts calcium phosphate cement (CPC) from application in vascularized bone tissue engineering. Our previous studies have developed a starch and BaSO4 incorporated calcium phosphate hybrid cement (CPHC) with improved mechanical strength and handling properties. However, the bioactivity-especially the angiogenic ability-is still absent and requires further improvement. Herein, based on the reported CPHC and the osteogenic and angiogenic properties of strontium (Sr) ions, a strontium-enhanced calcium phosphate hybrid cement (Sr-CPHC) was developed to improve both biological and physicochemical properties of CPC. Compared to CPC, the initial setting time of Sr-CPHC was prolonged from 2.2 min to 20.7 min. The compressive strength of Sr-CPHC improved from 11.21 MPa to 45.52 MPa compared with CPC as well. Sr-CPHC was biocompatible and showed promotion of alkaline phosphatase (ALP) activity, calcium nodule formation and osteogenic relative gene expression, suggesting high osteogenic-inductivity. Sr-CPHC also facilitated the migration and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro and up-regulated the expression of the vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1). In vivo evaluation showed marked new bone formation in a rat calvarial defect model with Sr-CPHC implanted. Sr-CPHC also exhibited enhancement of neovascularization in subcutaneous connective tissue in a rat subcutaneous implantation model. Thus, the Sr-CPHC with the dual effects of osteogenesis and angiogenesis shows great potential for clinical applications such as the repair of ischemic osteonecrosis and critical-size bone defects.

Phosphodiesterase-5 inhibition improves bone regeneration at the early stages of ischemic osteonecrosis of the femoral head in rats.

Posttraumatic osteonecrosis of the femoral head (ONFH) affects patients at different ages and may lead to functional limitation and joint replacement, with total hip arthroplasty, which is a costly procedure. Proposed methods to optimize ischemic tissue regeneration have been reported. Phosphodiesterase-5 inhibitors act by inhibiting the degradation of guanosine 3',5'-cyclic monophosphate in the nitric oxide pathway, increasing its bioavailability and promoting vascular endothelial growth factor (VEGF)-mediated neovascular recruitment and the induction of tissue regeneration in the traumatized bone. Thirty male Sprague-Dawley rats (6 months old) were subjected to an experimental model of traumatic ONFH divided into two groups, according to the administration of 5 mg/kg sildenafil or water (control group). Rats were then killed at 7, 14, and 21 days. Histological (Goldner's trichrome), histochemical (periodic acid-Schiff [PAS]), and immunohistochemical (VEGF and osteopontin [OPN]) techniques were used to quantify bone and vascular responses. Higher levels of VEGF (p < 0.01) and OPN (p < 0.01) immunostaining in the epiphysis, the greater formation of osteoid tissue (p < 0.01 on Day 7; p < 0.05 on Day 14), and higher levels of PAS staining (p < 0.01 on Day 7) were observed in the sildenafil-treated group. The present study demonstrated that sildenafil optimized bone tissue regeneration by increasing VEGF signaling and OPN expression, with increased bone formation (osteoid and carbohydrate macromolecule deposition) in the early stages following traumatic ischemic insult. Thus, sildenafil treatment may improve the prognosis of patients with osteonecrosis.

Elevation of Proinflammatory Cytokine HMGB1 in the Synovial Fluid of Patients With Legg-Calvé-Perthes Disease and Correlation With IL-6.

ABSTRACTLegg‐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.

Osteoblasts/Osteocytes sirtuin6 Is Vital to Preventing Ischemic Osteonecrosis Through Targeting VDR-RANKL Signaling.

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).

Anti-Interleukin-6 Therapy Decreases Hip Synovitis and Bone Resorption and Increases Bone Formation Following Ischemic Osteonecrosis of the Femoral Head.

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of ischemic femoral head osteonecrosis, which produces chronic hip synovitis, permanent femoral head deformity, and premature osteoarthritis. Currently, there is no medical therapy for LCPD. Interleukin-6 (IL-6) is significantly elevated in the synovial fluid of patients with LCPD. We hypothesize that IL-6 elevation promotes chronic hip synovitis and impairs bone healing after ischemic osteonecrosis. We set out to test if anti-IL-6 therapy using tocilizumab can decrease hip synovitis and improve bone healing in the piglet model of LCPD. Fourteen piglets were surgically induced with ischemic osteonecrosis and assigned to two groups: the no treatment group (n = 7) and the tocilizumab group (15 to 20 mg/kg, biweekly intravenous injection, n = 7). All animals were euthanized 8 weeks after the induction of osteonecrosis. Hip synovium and femoral heads were assessed for hip synovitis and bone healing using histology, micro-CT, and histomorphometry. The mean hip synovitis score and the number of synovial macrophages and vessels were significantly lower in the tocilizumab group compared with the no treatment group (p < .0001, p = .01, and p < .01, respectively). Micro-CT analysis of the femoral heads showed a significantly higher bone volume in the tocilizumab group compared with the no treatment group (p = .02). The histologic assessment revealed a significantly lower number of osteoclasts per bone surface (p < .001) in the tocilizumab group compared with the no treatment group. Moreover, fluorochrome labeling showed a significantly higher percent of mineralizing bone surface (p < .01), bone formation rate per bone surface (p < .01), and mineral apposition rate (p = .04) in the tocilizumab group. Taken together, tocilizumab therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. This study provides preclinical evidence that tocilizumab decreases synovitis and improves bone healing in a large animal model of LCPD. © 2020 American Society for Bone and Mineral Research (ASBMR).

Endoplasmic reticulum stress mediates osteocyte death under oxygen-glucose deprivation in vitro

As a vascularized organ, bone is known to be susceptible to ischemia. Ischemic osteonecrosis or skeletal unloading lead to ischemia in bone microenvironment that causes osteocytes to suffer hypoxia and nutrition deprivation. ObjectiveTo explore the effects of Oxygen-glucose deprivation (OGD) on osteocytes and the potential mechanism. MethodsOGD model was established in cultured MLO-Y4 cell. Cell damage, intracellular oxidative stress and cell apoptosis were detected at different OGD times (0, 2, 4, 8, 12, 24 h), and the changes in endoplasmic reticulum (ER) stress-related indicators were observed. Furthermore, cells were treated with 4-phenylbutyrate sodium (4-PBA) to inhibit ER stress, and cell damage and oxidative stress level were detected. ResultsThe cell viability under OGD exhibited a significantly reduced in a time-dependent manner, and the level of intracellular reactive oxygen species (ROS) were increased, cell apoptosis and ER stress was induced. Inhibition of ER stress can reduce cell death and intracellular ROS levels. ConclusionOur study demonstrated that ER stress regulates OGD-induced apoptotic cell death in MLO-Y4 cells via intracellular ROS.

Nouvelle technique mini-invasive pour éliminer le contenu de moelle osseuse nécrotique de l’os épiphysaire : étude préliminaire sur l’os cadavérique

IntroductionLegg–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. HypothesisWe 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 methodsTwenty-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. ResultsThe 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/conclusionThe 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 evidenceIII, in vitro comparative study.

Development of a novel minimally invasive technique to washout necrotic bone marrow content from epiphyseal bone: A preliminary cadaveric bone study

IntroductionLegg-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. HypothesisWe 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 methodsTwenty-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. ResultsThe 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/conclusionThe 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 evidenceIII, in vitro comparative study.