Background: The process of necroptosis mediated by tumor necrosis factor alpha (TNF-alpha) might play an important role in the onset and development of the osteonecrosis of the femoral head (ONFH). The dysfunctions of bone microvascular endothelial cells (BMECs) have been identified as an important part of pathological processes in the steroid-induced ONFH. An aptamer is a single-stranded DNA or RNA oligonucleotide sequence. Previous studies have designed or screened various aptamers that could bind to specific targets or receptors in order to block their effects.Objective: There are two main objectives in this study: 1) to establish a TNF-alpha-induced ONFH model in human BMECs in vitro, 2) to verify the effects of the TNF-alpha aptamer (AptTNF-alpha) on blocking TNF-alpha activity in the ONFH model.Methods: Clinical samples were collected for Hematoxylin and Eosin (HE) staining, immunohistochemistry and further BMEC isolation. After cell culture and identification, the cell viability of BMECs after incubation with TNF-alpha was assessed by Cell Counting Kit-8 (CCK8). The necroptosis of BMECs was detected by the TUNEL and Annexin V-FITC/PI staining. The attenuation of TNF-alpha cytotoxicity by AptTNF-alpha was evaluated by CCK8 at first. Then, the molecular mechanism was explored by the quantitative real-time polymerase chain reaction and western blotting.Results: The expression level of TNF-alpha was significantly up-regulated in bone tissues of ONFH patients. The identification of BMECs was verified by the high expressions of CD31 and vWF. Results from CCK8, TUNEL staining and Annexin V-FITC/PI assay demonstrated reduced cell viability and increased necroptosis of BMECs after TNF-alpha stimulation. Further investigations showed that TNF-alpha cytotoxicity could be attenuated by the AptTNF-alpha in a dose-dependent manner. Necroptosis mediated by TNF-alpha in the ONFH model was regulated by the receptor-interacting protein kinase 1 (RIPK1)/receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL) signalling pathway.Conclusion: We established a TNF-alpha-induced ONFH model in human BMECs in vitro. Our study also demonstrated that the AptTNF-alpha could protect BMECs from necroptosis by inhibiting the RIP1/RIP3/MLKL signalling pathway. The Translational Potential of this Article: The effective protection from cell necroptosis provided by the DNA aptamer demonstrated its translational potential as a new type of TNF-alpha inhibitor in clinical treatments for pa-tients with ONFH.

Studies have shown that high-energy focused extracorporeal shock wave therapy (HF-ESWT) has a certain therapeutic effect on glucocorticoid-induced osteonecrosis of the femoral head (ONFH). This study aimed to observe the efficacy and safety of HF-ESWT as a precautionary measure to reduce the probability of glucocorticoid-induced ONFH. A prospective randomized controlled trial was designed to evaluate whether HF-ESWT (Group A) can significantly prevent the incidence of glucocorticoid-induced ONFH relative to a control group without shockwave intervention (Group B). MRI was used to assess whether all participants experienced ONFH at 3, 6, and 12 months after the intervention. Continuous scoring was used to evaluate the intervention results: the 10-cm visual analog scale (VAS) was used to evaluate pain, and the hip Harris score (HHS) was used to evaluate the function of the hip joint. Any adverse events were recorded. 153 patients (89 females and 64 males) who had been allocated to group A (75 patients) or Group B (78 patients) were included in the final analysis. The patients were 45.0 ​± ​13.0 years old. There were significant differences between the two groups in MRI diagnosis of ONFH patients (2 cases in Group A, 9 cases in Group B; p ​= ​0.034). Significant differences between groups were found in bilateral hip function measured using the HHS at 6 months (Left p ​= ​0.026; Right p ​= ​0.033) and 12 months (Left p ​= ​0.018; Right p ​= ​0.038). However, there was no difference in the functional results measured at 3 months and the VAS at any points. This study confirms that HF-ESWT can be successfully used to reduce the probability of glucocorticoid-induced ONFH. Pain and hip dysfunction are common clinical manifestations when ONFH is unavoidable. Therefore, HF-ESWT can be recommended for the prevention and intervention of ONFH high-risk populations receiving high-dose glucocorticoid therapy. The effective prevention of HF-ESWT on ONFH after high-dose glucocorticoid application demonstrated its transformation potential as a preventive method in the clinical prevention of glucocorticoid-induced ONFH.

Background. Multiple reports have demonstrated the therapeutic potential of extracorporeal shock wave (ESWT) in osteonecrosis of the femoral head (ONFH). However, few studies reported the changes in hip articular cartilage after the intervention. This study aimed to investigate the effect of ESWT on femoral head cartilage using a novel technique, quantitative T2‐mapping magnetic resonance imaging. Methods. A total of 143 eligible patients with unilateral early‐stage ONFH were randomized into the ESWT group and control group. Seventy‐three patients in the ESWT group received two sessions of ESWT with oral drug treatment, while seventy patients in the control group received oral drug treatment only. The visual analog pain scale (VAS) and Harris hip score (HHS) at 3‐month, 6‐month, and 12‐month follow‐up were used as the clinical evaluation index. The radiological evaluation index used the T2 mapping values, necrotic size, and China‐Japan Friendship Hospital (CJFH) classification. Results. A total of 143 patients (62 females and 81 males) were finally included, and the characteristics before treatment were comparable between the two groups. At the last follow‐up (12 months), the T2 values and ΔT2 changes in the ESWT group were all smaller than those in the control group (p = 0.042; p = 0.039), while the CJFH classification of ONFH and necrotic lesion size were not statistically significant. At 3 months and 6 months, the VAS in the ESWT group was lower than that in the control group (p = 0.021; p = 0.046) and the HHS in the ESWT group was higher (p = 0.028; p = 0.039). However, there were no significant differences in the VAS and HHS at 12 months between the ESWT and control groups. Conclusions. The results of the current study indicated that, based on drug treatment, ESWT is an effective treatment method for nontraumatic ONFH, which could result in significant pain relief and function restoration. Furthermore, it could delay the injury of femoral head cartilage during the progression of ONFH.

Background: Despite great efforts to develop microvascular bone chips in previous studies, current bone chips still lacked multi-component of human-derived cells close to human bone tissue. Bone microvascular endothelial cells (BMECs) were demonstrated to be closely related to the glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH). Tumor necrosis factor-alpha (TNF-α) aptamer has been proved to bind to its receptor and block cascade activities.Objective: There are two main objectives in this study: 1) to establish a multi-component bone-on-a-chip within the microfluidic system in vitro, 2) to explore the therapeutic potential of TNF-α aptamer on BMECs in the GC-induced ONFH model.Methods: Histological features of clinical samples were analyzed before BMECs isolation. The functional bone-on-a-chip consists of the vascular channel, stromal channel and structure channel. GC-induced ONFH model was established based on the multi-component of human-derived cells. Truncation and dimerization were performed on a previously reported DNA aptamer (VR11). BMECs apoptosis, cytoskeleton and angiogenesis status in the ONFH model were observed by the TUNEL staining and confocal microscope.Results: The multi-component of BMECs, human embryonic lung fibroblasts and hydroxyapatite were cultured within the microfluidic bone-on-a-chip. TNF-α was found up-regulated in the necrotic regions of femoral heads in clinical samples and similar results were re-confirmed in the ONFH model established in the microfluidic platform by detecting cell metabolites. Molecular docking simulations indicated that the truncated TNF-α aptamer could improve the aptamer-protein interactions. Further results from the TUNEL staining and confocal microscopy showed that the truncated aptamer could protect BMECs from apoptosis and alleviate GC-induced damages to cytoskeleton and vascularization.Conclusion: In summary, a microfluidic multi-component bone-on-a-chip was established with ‘off-chip’ analysis of cell metabolism. GC-induced ONFH model was achieved based on the platform. Our findings provided initial evidence on the possible potentials of TNF-α aptamer as a new type of TNF-α inhibitor for patients with ONFH.

Background: Phase-contrast imaging (PCI) with synchrotron hard X-ray was used to observe the changes in bone tissue morphology and microstructure in rabbit models of early glucocorticoid-induced osteonecrosis of the femoral head (ONFH), and to evaluate the intervention effect of Icariin.Methods: Fifty mature New Zealand rabbits (weighing 2.5–3.0 kg) were randomly divided into a control group (n = 10), a glucocorticoid group (n = 20), and an Icariin group (n = 20). The glucocorticoid group and the Icariin group were sequentially injected with lipopolysaccharide (LPS) and methylprednisolone (MPS) to establish a glucocorticoid-induced ONFH animal model. The Icariin group was given Icariin solution when methylprednisolone was injected for the first time, and the control group and glucocorticoid group were given the same amount of normal saline. Animals were sacrificed after 6 weeks, and bilateral femoral head specimens were taken for research. The right femoral head was observed by PCI with synchrotron hard X-ray technology, and the left femoral head was verified by Micro-CT scanning and HE staining.Results: Forty-three animals (nine in the control group, sixteen in the glucocorticoid group, and eighteen in the Icariin group) were included in the study. PCI with synchrotron hard X-ray revealed that the trabecular bone in the glucocorticoid group was thinned, broken, and structurally damaged, whereas the trabecular bone in the Icariin group had normal volume, thickness, and a relatively intact structure. Micro-CT scan reconstruction and HE staining were used to verify the reliability of this technique in identifying osteonecrosis.Conclusion: The effects of Icariin were observed in an early glucocorticoid-induced ONFH rabbit model using PCI with synchrotron hard X-ray. Icariin weakens the destructive effect of glucocorticoids on bone tissue structure, improves bone tissue morphology, and stabilizes bone microstructure. This technique may provide a definitive, non-invasive alternative to histological examination for the diagnosis of early ONFH.

Background: This study aimed to develop a nomogram capable of predicting the probability of femoral head collapse based on an 18-year follow-up cohort of convalescent severe acute respiratory syndrome (SARS) patients with glucocorticoid-induced osteonecrosis of the femoral head (ONFH).Methods: Data on the natural history of 120 patients (205 hips) who underwent glucocorticoid-induced ONFH at China-Japan Friendship Hospital (CJFH) in 2003 were retrospectively collected. Follow-up was conducted from June 2003 to October 2021. A nomogram was developed in a training cohort and validated in another cohort.Results: A total of 205 hips were included for analysis, with 143 hips in the training cohorts and 62 hips in the validation cohorts. After 18 years of follow-up, 53 femoral heads collapsed, while 152 femoral heads spontaneously repaired to some extent (necrotic areas reduced or vanished). Following multivariate regression analysis, the Association Research Circulation Osseous (ARCO) staging, necrosis index (NI), and CJFH Classification were entered into the nomogram. The nomogram showed robust discrimination, with an AUC of 0.907 (95% CI: 0.85–0.96). The calibration curves showed an agreement between the probability as predicted by the nomogram and the actual probability. Application of the nomogram in the validation cohort also yielded good discrimination (AUC, 0.876, 95% CI: 0.7751–0.9761) and calibration.Conclusion: The nomogram successfully predicted femoral head collapse in glucocorticoid-induced ONFH. With the nomogram, the prognosis for an individual patient with glucocorticoid-induced ONFH can be determined, which can lead to a rational therapeutic choice.