Luteolin ameliorates necroptosis in Glucocorticoid-induced osteonecrosis of the femoral head via RIPK1/RIPK3/MLKL pathway based on network pharmacology analysis.

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is deeply relevant to damage and dysfunction of bone microvascular endothelial cells (BMECs). Recently, necroptosis, a newly programmed cell death with necrotic appearance, has garnered increasing attention. Luteolin, a flavonoid compound derived from Rhizoma Drynariae, has numerous pharmacological properties. However, the effect of Luteolin on BMECs in GIONFH through the necroptosis pathway has not been extensively investigated. Based on network pharmacology analysis, 23 genes were identified as potential targets for the therapeutic effect of Luteolin in GIONFH via the necroptosis pathway, with RIPK1, RIPK3, and MLKL being the hub genes. Immunofluorescence staining results revealed high expression of vWF and CD31 in BMECs. In vitro experiments showed that incubation with dexamethasone led to reduced proliferation, migration, angiogenesis ability, and increased necroptosis of BMECs. However, pretreatment with Luteolin attenuated this effect. Based on molecular docking analysis, Luteolin exhibited strong binding affinity with MLKL, RIPK1, and RIPK3. Western blotting was utilized to detect the expression of p-MLKL, MLKL, p-RIPK3, RIPK3, p-RIPK1, and RIPK1. Intervention with dexamethasone resulted in a significant increase in the p-RIPK1/RIPK1 ratio, but the effects of dexamethasone were effectively counteracted by Luteolin. Similar findings were observed for the p-RIPK3/RIPK3 ratio and the p-MLKL/MLKL ratio, as anticipated. Therefore, this study demonstrates that Luteolin can reduce dexamethasone-induced necroptosis in BMECs via the RIPK1/RIPK3/MLKL pathway. These findings provide new insights into the mechanisms underlying the therapeutic effects of Luteolin in GIONFH treatment. Additionally, inhibiting necroptosis could be a promising novel approach for GIONFH therapy.

Aspirin prevents estrogen deficiency-induced bone loss by inhibiting osteoclastogenesis and promoting osteogenesis.

BACKGROUND: Aspirin is a commonly used antipyretic, analgesic, and anti-inflammatory drug. Numerous researches have demonstrated that aspirin exerts multiple biological effects on bone metabolism. However, its spatiotemporal roles remain controversial according to the specific therapeutic doses used for different clinical conditions, and the detailed mechanisms have not been fully elucidated. Hence, in the present study, we aimed to identify the dual effects of different aspirin dosages on osteoclastic activity and osteoblastic bone formation in vitro and in vivo. METHODS: The effects of varying doses of aspirin on osteoclast and osteoblast differentiation were evaluated in vitro. The underlying molecular mechanisms were detected using quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence techniques. An ovariectomized rat osteoporosis model was used to assess the bone-protective effects of aspirin in vivo. RESULTS: Aspirin dose-dependently suppressed RANKL-induced osteoclasts differentiation and bone resorption in vitro and reduced the expression of osteoclastic marker genes, including TRAP, cathepsin K, and CTR. Further molecular analysis revealed that aspirin impaired the RANKL-induced NF-κB and MAPK signaling pathways and prevented the nuclear translocation of the NF-κB p65 subunit. Low-dose aspirin promoted osteogenic differentiation, whereas these effects were attenuated when high-dose aspirin was administered. Both low and high doses of aspirin prevented bone loss in an ovariectomized rat osteoporosis model in vivo. CONCLUSION: Aspirin inhibits RANKL-induced osteoclastogenesis and promotes osteogenesis in a dual regulatory manner, thus preventing bone loss in vivo. These data indicate that aspirin has potential applications in the prevention and treatment of osteopenia.

The protective effect of DNA aptamer on osteonecrosis of the femoral head by alleviating TNF-α-mediated necroptosis via RIP1/RIP3/MLKL pathway.

Background: The process of necroptosis mediated by tumor necrosis factor alpha (TNF-α) 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-α -induced ONFH model in human BMECs in vitro, 2) to verify the effects of the TNF-α aptamer (AptTNF-α) on blocking TNF-α 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-α 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-α cytotoxicity by AptTNF-α 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-α 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-α stimulation. Further investigations showed that TNF-α cytotoxicity could be attenuated by the AptTNF-α in a dose-dependent manner. Necroptosis mediated by TNF-α 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-α-induced ONFH model in human BMECs in vitro. Our study also demonstrated that the AptTNF-α 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-α inhibitor in clinical treatments for patients with ONFH.

Verification and clinical translation of a newly designed "Skywalker" robot for total knee arthroplasty: A prospective clinical study.

OBJECTIVE: To evaluate accuracy of an innovative "Skywalker" system, a newly designed, robot-assisted operation system for orthopaedics via a clinical trial at knee joint. METHODS: We conducted a prospective analysis of the clinical data of 31 patients who underwent total knee arthroplasty assisted by the "Skywalker" robot (Microport, Suzhou, China) from June 2020 to January 2021. Five male patients and 26 female patients aged 69.68 â€‹± â€‹6.11 years (range: 57-79 years) were diagnosed with knee osteoarthritis and indicated for surgery. The "Skywalker" surgical robotic system was adopted to make a preoperative plan for knee arthroplasty. When the robotic arm reached the specified position during the operation, a single surgeon performed the osteotomy with a cutting saw through the cutting jig, and the difference between the actual and the expected resection thickness, and the preoperative and postoperative lower limb alignments were measured. RESULTS: The actual error of the resection thickness was the difference between the actual and the expected resection thickness. The absolute error of the resection thickness was the absolute value of the actual error of resection thickness. The absolute errors of the resection thickness of the medial and lateral condyle of the distal femur, the medial and lateral posterior condyle of the femur, and the medial and lateral sides of the tibial plateau were 0.87 â€‹± â€‹0.63 â€‹mm, 1.02 â€‹± â€‹0.67 â€‹mm, 0.74 â€‹± â€‹0.46 â€‹mm, 0.98 â€‹± â€‹0.81 â€‹mm, 0.92 â€‹± â€‹0.66 â€‹mm, and 1.04 â€‹± â€‹0.84 â€‹mm, respectively. The absolute angle errors between the actual postoperative angles and the preoperative planned angles of the lower limb alignment angles, coronal femoral component angles, and coronal tibial component angles were 1.46° â€‹± â€‹0.95°, 1.13° â€‹± â€‹1.01°, and 1.05° â€‹± â€‹0.73°, respectively. Besides, 100% of the absolute error of the HKA angles was within 3°. In addition, compared to the preoperative lower limb alignment angle, 90.32% of the postoperative lower limb alignment angles of 31 patients were closer to 180° after the operation. All 31 patients underwent a successful surgery, and no relevant complications occurred after the operation, such as surgical site infection, deep venous thrombosis, or vascular and nerve injury. CONCLUSION: The "Skywalker" system has good osteotomy accuracy, can achieve the planned angles well, and is expected to assist surgeons in performing accurate bone cuts and reconstructing planned lower limb alignments in the relevant clinical applications in future.

'Skywalker' surgical robot for total knee arthroplasty: An experimental sawbone study.

BACKGROUND: Currently, robot-assisted surgical systems are used to reduce the error range of total knee arthroplasty (TKA) osteotomy and component positioning. METHODS: We used 20 sawbone models of the femur and 20 sawbone models of the tibia and fibula to evaluate the osteotomy effect of 'Skywalker' robot-assisted TKA. RESULTS: The maximal movement of the cutting jig was less than 0.25 mm at each osteotomy plane. The mean and standard deviation values of the angle deviation between the planned osteotomy plane and the actual osteotomy plane at each osteotomy plane were not more than 1.03° and 0.55°, respectively. The mean and standard deviation values of absolute error of resection thickness at each osteotomy position were less than 0.78 and 0.71 mm, respectively. CONCLUSIONS: The 'Skywalker' system has good osteotomy accuracy, can achieve the planned osteotomy well and is expected to assist surgeons in performing accurate TKA in clinical applications in future.

TiO2 nanotubes regulate histone acetylation through F-actin to induce the osteogenic differentiation of BMSCs.

Bone integration on the surface of titanium prosthesis is critical to the success of implant surgery. Good Bone integration at the contact interface is the basis of long-term stability. TiO2 nanotubes have become one of the most commonly used modification techniques for artificial joint prostheses and bone defect implants due to their good biocompatibility, mechanical properties and chemical stability. TiO2 nanotubes can promote F-actin polymerization in bone mesenchymal stem cells (BMSCs) and osteogenic differentiation. The possibility of F-actin as an upstream part to regulate GCN5 initiation of osteogenesis was discussed. The results of gene loss and functional acquisition assay, immunoblotting assay and fluorescence staining assay showed that TiO2 nanotubes could promote the differentiation of BMSCs into osteoblasts. The intervention of TiO2 nanotubes can make BMSCs form stronger F-actin fibre bundles, which can drive the differentiation process of osteogenesis. Our results showed that F-actin mediated nanotube-induced cell differentiation through promoting the expression of GCN5 and enhancing the function of GCN5 and GCN5 was a key regulator of the osteogenic differentiation of BMSCs induced by TiO2 nanotubes as a downstream mediated osteogenesis of F-actin, providing a novel insight into the study of osteogenic differentiation on surface of TiO2 nanotubes.

F-actin Regulates Osteoblastic Differentiation of Mesenchymal Stem Cells on TiO2 Nanotubes Through MKL1 and YAP/TAZ.

Titanium and titanium alloys are widely used in orthopedic implants. Modifying the nanotopography provides a new strategy to improve osseointegration of titanium substrates. Filamentous actin (F-actin) polymerization, as a mechanical loading structure, is generally considered to be involved in cell migration, endocytosis, cell division, and cell shape maintenance. Whether F-actin is involved and how it functions in nanotube-induced osteogenic differentiation of mesenchymal stem cells (MSCs) remain to be elucidated. In this study, we fabricated TiO2 nanotubes on the surface of a titanium substrate by anodic oxidation and characterized their features by scanning electron microscopy (SEM), X-ray energy dispersive analysis (EDS), and atomic force microscopy (AFM). Alkaline phosphatase (ALP) staining, Western blotting, qRT-PCR, and immunofluorescence staining were performed to explore the osteogenic potential, the level of F-actin, and the expression of MKL1 and YAP/TAZ. Our results showed that the inner diameter and roughness of TiO2 nanotubes increased with the increase of the anodic oxidation voltage from 30 to 70 V, while their height was 2 µm consistently. Further, the larger the tube diameter, the stronger the ability of TiO2 nanotubes to promote osteogenic differentiation of MSCs. Inhibiting F-actin polymerization by Cyto D inhibited osteogenic differentiation of MSCs as well as the expression of proteins contained in focal adhesion complexes such as vinculin (VCL) and focal adhesion kinase (FAK). In contrast, after Jasp treatment, polymerization of F-actin enhanced the expression of RhoA and transcription factors YAP/TAZ. Based on these data, we concluded that TiO2 nanotubes facilitated the osteogenic differentiation of MSCs, and this ability was enhanced with the increasing diameter of the nanotubes within a certain range (30-70 V). F-actin mediated this process through MKL1 and YAP/TAZ.

Clinical Applications of 3-Dimensional Printing Technology in Hip Joint.

Three-dimensional (3D) printing is a digital rapid prototyping technology based on a discrete and heap-forming principle. We identified 53 articles from PubMed by searching "Hip" and "Printing, Three-Dimensional"; 52 of the articles were published from 2015 onwards and were, therefore, initially considered and discussed. Clinical application of the 3D printing technique in the hip joint mainly includes three aspects: a 3D-printed bony 1:1 scale model, a custom prosthesis, and patient-specific instruments (PSI). Compared with 2-dimensional image, the shape of bone can be obtained more directly from a 1:1 scale model, which may be beneficial for preoperative evaluation and surgical planning. Custom prostheses can be devised on the basis of radiological images, to not only eliminate the fissure between the prosthesis and the patient's bone but also potentially resulting in the 3D-printed prosthesis functioning better. As an alternative support to intraoperative computer navigation, PSI can anchor to a specially appointed position on the patient's bone to make accurate bone cuts during surgery following a precise design preoperatively. The 3D printing technique could improve the surgeon's efficiency in the operating room, shorten operative times, and reduce exposure to radiation. Well known for its customization, 3D printing technology presents new potential for treating complex hip joint disease.

Mechanical strain promotes osteogenic differentiation of mesenchymal stem cells on TiO2 nanotubes substrate.

Previous studies demonstrated cycle mechanical strain induced osteogenic differentiation of MSCs. But in general, MSCs are typically seeded on a flexible membrane or within a soft matrix. TiO2 nanotubes substrate topography plays a critical role in promoting the MSCs response and affects MSCs fate. Titanium implants surface modified by TiO2 nanotubes topography provides the opportunity to improve osseointegration by additionally regulating the MSCs fate. Titanium is one of most commonly used materials in the orthopedics and can undergo elastic deformation under certain mechanical stress. Therefore, for clinic trails, it is necessary to investigate the effect of mechanical strain on osteogenesis of MSCs on TiO2 nanotubes modified titanium substrate. But until now, there has been no research focused on the relationship between mechanical strain and osteogenesis of MSCs on the TiO2 nanotubes topography substrate. Here, we firstly applied the mechanical stress to the TiO2 nanotubes modified titanium specimen to investigate the effects of mechanical strain on the biological behaviors of MSCs. Our present study showed that mechanical strain promoted cell proliferation, spreading and increased vinculin expression of MSCs on the TiO2 nanotubes substrate. Additionally, mechanical strain enhanced the ALP activity and osteogenesis genes expression such as Runx2, BSP, ALP, OPN and OCN. Our results preliminarily demonstrated that mechanical strain enhanced the osteogenic differentiation of MSCs through the FAK-Erk1/2-Runx2 pathway on the TiO2 nanotubes substrate.