Picein alleviates oxidative stress and promotes bone regeneration in osteoporotic bone defect by inhibiting ferroptosis via Nrf2/HO-1/GPX4 pathway.

Osteoporosis (OP) can result in slower bone regeneration than the normal condition due to abnormal oxidative stress and high levels of reactive oxygen species (ROS), a condition detrimental for bone formation, making the OP-related bone healing a significant clinical challenge. As the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) is closely related to bone regeneration; currently, this study assessed the effects of Picein on BMSCs in vitro and bone regeneration in osteoporotic bone defect in vivo. Cell viability was determined by CCK-8 assay. The production of (ROS), malonaldehyde, superoxide dismutase activities, and glutathione was evaluated by using commercially available kits, and a flow cytometry analysis was adopted to detect macrophage polarization. Osteogenic capacity of BMSCs was evaluated by alkaline phosphatase (ALP) activity, ALP staining, and Alizarin red S staining. The expression of osteogenic-related proteins (OPN, Runx-2, OCN) and osteogenic-related genes (ALP, BMP-4, COL-1, and Osterix) were evaluated by Western blotting and real-time PCR (RT-PCR). In addition, proliferation, migration ability, and angiogenic capacity of human umbilical vein endothelial cells (HUVECs) were evaluated by EdU staining, scratch test, transwell assay, and tube formation assay, respectively. Angiogenic-related genes (VEGF, vWF, CD31) were also evaluated by RT-PCR. Results showed that Picein alleviated erastin-induced oxidative stress, enhanced osteogenic differentiation capacity of BMSCs, angiogenesis of HUVECs, and protects cells against ferroptosis through Nrf2/HO-1/GPX4 axis. Moreover, Picein regulate immune microenvironment by promoting the polarization of M2 macrophages in vitro. In addition, Picein also reduce the inflammation levels and promotes bone regeneration in osteoporotic bone defect in OP rat models in vivo. Altogether, these results suggested that Picein can promote bone regeneration and alleviate oxidative stress via Nrf2/HO-1/GPX4 pathway, offering Picein as a novel antioxidant agent for treating osteoporotic bone defect.

Injectable, anti-collapse, adhesive, plastic and bioactive bone graft substitute promotes bone regeneration by moderating oxidative stress in osteoporotic bone defect.

The treatment of osteoporotic bone defect remains a big clinical challenge because osteoporosis (OP) is associated with oxidative stress and high levels of reactive oxygen species (ROS), a condition detrimental for bone formation. Anti-oxidative nanomaterials such as selenium nanoparticles (SeNPs) have positive effect on osteogenesis owing to their pleiotropic pharmacological activity which can exert anti-oxidative stress functions to prevent bone loss and facilitate bone regeneration in OP. In the current study a strategy of one-pot method by introducing Poly (lactic acid-carbonate) (PDT) and ß-Tricalcium Phosphate (ß-TCP) with SeNPs, is developed to prepare an injectable, anti-collapse, shape-adaptive and adhesive bone graft substitute material (PDT-TCP-SE). The PDT-TCP-SE bone graft substitute exhibits sufficient adhesion in biological microenvironments and osteoinductive activity, angiogenic effect and anti-inflammatory as well as anti-oxidative effect in vitro and in vivo. Moreover, the PDT-TCP-SE can protect BMSCs from erastin-induced ferroptosis through the Sirt1/Nrf2/GPX4 antioxidant pathway, which, in together, demonstrated the bone graft substitute material as an emerging biomaterial with potential clinical application for the future treatment of osteoporotic bone defect. STATEMENT OF SIGNIFICANCE: Injectable, anti-collapse, adhesive, plastic and bioactive bone graft substitute was successfully synthesized. Incorporation of SeNPs with PDT into ß-TCP regenerated new bone in-situ by moderating oxidative stress in osteoporotic bone defects area. The PDT-TCP-SE bone graft substitute reduced high ROS levels in osteoporotic bone defect microenvironment. The bone graft substitute could also moderate oxidative stress and inhibit ferroptosis via Sirt1/Nrf2/GPX4 pathway in vitro. Moreover, the PDT-TCP-SE bone graft substitute could alleviate the inflammatory environment and promote bone regeneration in osteoporotic bone defect in vivo. This biomaterial has the advantages of simple synthesis, biocompatibility, anti-collapse, injectable, and regulation of oxidative stress level, which has potential application value in bone tissue engineering.

Absorbable calcium and phosphorus bioactive membranes promote bone marrow mesenchymal stem cells osteogenic differentiation for bone regeneration.

Large segmental bone defects are commonly operated with autologous bone grafting, which has limited bone sources and poses additional surgical risks. In this study, we fabricated poly(lactide-co-glycolic acid) (PLGA)/ß-tricalcium phosphate (ß-TCP) composite membranes by electrostatic spinning and further promoted osteogenesis by regulating the release of ß-TCP in the hope of replacing autologous bone grafts in the clinical practice. The addition of ß-TCP improved the mechanical strength of PLGA by 2.55 times. Moreover, ß-TCP could accelerate the degradation of PLGA and neutralize the negative effects of acidification of the microenvironment caused by PLGA degradation. In vitro experiments revealed that PLGA/TCP10 membranes are biocompatible and the released ß-TCP can modulate the activity of osteoblasts by enhancing the calcium ions concentration in the damaged area and regulating the pH of the local microenvironment. Simultaneously, an increase in ß-TCP can moderate the lactate content of the local microenvironment, synergistically enhancing osteogenesis by promoting the tube-forming effect of human umbilical vein endothelial cells. Therefore, it is potential to utilize PLGA/TCP bioactive membranes to modulate the microenvironment at the site of bone defects to promote bone regeneration.

Remodeling of the Intra-Conduit Inflammatory Microenvironment to Improve Peripheral Nerve Regeneration with a Neuromechanical Matching Protein-Based Conduit.

Peripheral nerve injury (PNI) remains a challenging area in regenerative medicine. Nerve guide conduit (NGC) transplantation is a common treatment for PNI, but the prognosis of NGC treatment is unsatisfactory due to 1) neuromechanical unmatching and 2) the intra-conduit inflammatory microenvironment (IME) resulting from Schwann cell pyroptosis and inflammatory-polarized macrophages. A neuromechanically matched NGC composed of regenerated silk fibroin (RSF) loaded with poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) (P:P) and dimethyl fumarate (DMF) are designed, which exhibits a matched elastic modulus (25.1 ± 3.5 MPa) for the peripheral nerve and the highest 80% elongation at break, better than most protein-based conduits. Moreover, the NGC can gradually regulate the intra-conduit IME by releasing DMF and monitoring sciatic nerve movements via piezoresistive sensing. The combination of NGC and electrical stimulation modulates the IME to support PNI regeneration by synergistically inhibiting Schwann cell pyroptosis and reducing inflammatory factor release, shifting macrophage polarization from the inflammatory M1 phenotype to the tissue regenerative M2 phenotype and resulting in functional recovery of neurons. In a rat sciatic nerve crush model, NGC promoted remyelination and functional and structural regeneration. Generally, the DMF/RSF/P:P conduit provides a new potential therapeutic approach to promote nerve repair in future clinical treatments.

Rab32 facilitates Schwann cell pyroptosis in rats following peripheral nerve injury by elevating ROS levels.

BACKGROUND: Peripheral nerve injury (PNI) is commonly observed in clinical practice, yet the underlying mechanisms remain unclear. This study investigated the correlation between the expression of a Ras-related protein Rab32 and pyroptosis in rats following PNI, and potential mechanisms have been explored by which Rab32 may influence Schwann cells pyroptosis and ultimately peripheral nerve regeneration (PNR) through the regulation of Reactive oxygen species (ROS) levels. METHODS: The authors investigated the induction of Schwann cell pyroptosis and the elevated expression of Rab32 in a rat model of PNI. In vitro experiments revealed an upregulation of Rab32 during Schwann cell pyroptosis. Furthermore, the effect of Rab32 on the level of ROS in mitochondria in pyroptosis model has also been studied. Finally, the effects of knocking down the Rab32 gene on PNR were assessed, morphology, sensory and motor functions of sciatic nerves, electrophysiology and immunohistochemical analysis were conducted to assess the therapeutic efficacy. RESULTS: Silencing Rab32 attenuated PNI-induced Schwann cell pyroptosis and promoted peripheral nerve regeneration. Furthermore, our findings demonstrated that Rab32 induces significant oxidative stress by damaging the mitochondria of Schwann cells in the pyroptosis model in vitro. CONCLUSION: Rab32 exacerbated Schwann cell pyroptosis in PNI model, leading to delayed peripheral nerve regeneration. Rab32 can be a potential target for future therapeutic strategy in the treatment of peripheral nerve injuries.

Robust Osteoconductive ß-Tricalcium Phosphate/L-poly(lactic acid) Membrane via Orientation-Strengthening Technology.

L-poly(lactic acid) (PLLA) is a biodegradable material with multiple biomedical application potentials, especially as a membrane for guided bone regeneration. In terms of its low strength and poor osteogenic activity, improving these two properties is the key to resolve the limitations of PLLA for bone-associated applications. Herein, an orientation-strengthening technology (OST) was developed to reinforce PLLA's mechanical strength by introducing biocompatible ß-tricalcium phosphate (ß-TCP) to improve the crystallinity of PLLA, allowing for the formation of a highly oriented architecture to acquire an advanced membrane with high mechanical property. Furthermore, the addition of ß-TCP nanoparticles significantly promotes the osteogenic activity of the composites. The tensile strength of the membrane containing 5 wt % ß-TCP was 220 MPa, which was 4-folds that of the native polylactic acid fabricated via the conventional method. The oriented microstructure enhanced both the mechanical strength and the osteogenic activity of the material. The parallel grooves on the material surface are similar to the mineralized collagen fibers on the bone surface, which promoted the growth and differentiation of osteoblasts, with ß-TCP further contributing to the osteoconductive effect. The combination of ß-TCP and orientation-strengthening effect endows the material with higher mechanical properties and bioactivities, which provides an advanced manufacturing strategy for the preparation of PLLA-based materials for bone repair.

Punicalagin attenuates TNF-α-induced oxidative damage and promotes osteogenic differentiation of bone mesenchymal stem cells by activating the Nrf2/HO-1 pathway.

Oxidative stress is one of the most important factors in changing bone homeostasis. Redox homeostasis plays a key role in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and the angiogenesis ability of human umbilical vein endothelial cells (HUVECs) for bone regeneration. Currently, this study assessed the effects of punicalagin (PUN) on BMSCs and HUVECs. Cell viability was determined by CCK-8 assay. A flow cytometry analysis was adopted to detect macrophage polarization. The production of reactive oxygen stress (ROS), glutathione (GSH), malonaldehyde (MDA) and superoxide dismutase (SOD) activities were evaluated by using commercially-available kits. Osteogenic capacity of BMSCs was evaluated by ALP activity, ALP staining and ARS staining. The expression of osteogenic-related proteins (OCN, Runx-2, OPN) and Nrf/HO-1 levles were evaluated by Western blotting. Osteogenic-related genes (Osterix, COL-1, BMP-4, ALP) were evaluated by RT-PCR. Migration ability and invasion ability of HUVECs were evaluated by wound healing assay and Transwell assay. Angiogenic ability was detected by tube formation assay and the expression of angiogenic-related genes (VEGF, vWF, CD31) were evaluated by RT-PCR. Results showed that PUN alleviated oxidative stress by TNF-α, enhanced osteogenic differentiation in BMSCs and angiogenesis in HUVECs. Moreover, PUN regulate immune microenvironment by promoting the polarization of M2 macrophages and reduce the oxidative stress related products by activating Nrf2/HO-1 pathway. Altogether, these results suggested that PUN can promote osteogenic capacity of BMSCs, angiogenesis of HUVECs, alleviate oxidative stress via Nrf2/HO-1 pathway, offering PUN as a novel antioxidant agent for treating bone loss diseases.

Inhibition of Schwann Cell Pyroptosis Promotes Nerve Regeneration in Peripheral Nerve Injury in Rats.

Background: Peripheral nerve injury (PNI) is one of the most debilitating injuries, but therapies for PNI are still far from satisfactory. Pyroptosis, a recently identified form of cell death, has been demonstrated to participate in different diseases. However, the role of pyroptosis of Schwann cells in PNI remains unclear. Methods: We established a rat PNI model, and western blotting, transmission electron microscopy, and immunofluorescence staining were used to confirm pyroptosis of Schwann cells in PNI in vivo. In vitro, pyroptosis of Schwann cells was induced by lipopolysaccharides (LPS)+adenosine triphosphate disodium (ATP). An irreversible inhibitor of pyroptosis, acetyl (Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-cmk), was used to attenuate Schwann cell pyroptosis. Moreover, the influence of pyroptotic Schwann cells on the function of dorsal root ganglion neurons (DRGns) was analyzed by a coculture system. Finally, the rat PNI model was intraperitoneally treated with Ac-YVAD-cmk to observe the effect of pyroptosis on nerve regeneration and motor function. Results: Schwann cell pyroptosis was notably observed in the injured sciatic nerve. LPS+ATP treatment effectively induced Schwann cell pyroptosis, which was largely attenuated by Ac-YVAD-cmk. Additionally, pyroptotic Schwann cells inhibited the function of DRGns by secreting inflammatory factors. A decrease in pyroptosis in Schwann cells promoted regeneration of the sciatic nerve and recovery of motor function in rats. Conclusion: Given the role of Schwann cell pyroptosis in PNI progression, inhibition of Schwann cell pyroptosis might be a potential therapeutic strategy for PNI in the future.

Engeletin alleviates erastin-induced oxidative stress and protects against ferroptosis via Nrf2/Keap1 pathway in bone marrow mesenchymal stem cells.

Ferroptosis is a novel form of cell death, which is a unique modality of cell death and closely associated with iron concentrations, generation of reactive oxygen species (ROS), and accumulation of the lipid reactive oxygen species. In the present study, the anti-ferroptosis effects of Engeletin was studied in erastin-induced bone marrow mesenchymal stem cells (BMSCs). After treatment with Engeletin, cell viability was determined by CCK-8 assay. The production of ROS, malonaldehyde (MDA), Superoxide dismutase (SOD) activities and glutathione peroxidase (GSH) were detected by using commercially-available kits. Ferroptosis-related proteins (GPX4, SLC7A11, TFR1, FPN1, Nrf2, Keap1) were evaluated by Western blotting. Osteogenic capacity was evaluated by ALP staining and ARS staining. The expression of osteogenic-related proteins (OPN, Runx2, OCN) were evaluated by Western blotting and changes in mRNA (ALP, BMP-2, COL-1, Osterix) were evaluated by RT-PCR. Consistent improvements in angiogenesis are observed with Engeletin in the presence of erastin. Engeletin significantly alleviated erastin-induced oxidative damage and protected against ferroptosis in BMSCs. Ferroptosis was inhibited by Engeletin, leading to decreasing reducing accumulation of ROS and lipid peroxidation products. Moreover, Engeletin promoted osteogenic differentiation in BMSCs and angiogenesis in human umbilical vein endothelial cells (HUVECs). Taken together, these findings indicate that Engeletin can protect BMSCs from erastin-induced ferroptosis through the Nrf2/Keap1 antioxidant pathway and identify Engeletin as a novel ferroptosis inhibitor, suggesting that Engeletin may promote resistance to ferroptosis and enable osteogenic function of BMSCs.

Polydopamine-Decorated PLCL Conduit to Induce Synergetic Effect of Electrical Stimulation and Topological Morphology for Peripheral Nerve Regeneration.

Due to the limited self-repairing capacity after peripheral nerve injuries (PNI), artificial nerve conduits are widely applied to facilitate neural regeneration. Exogenous electrical stimulation (ES) that is carried out by the conductive conduit regulates the biological behavior of Schwann cells (SCs). Meanwhile, a longitudinal surface structure counts to guide axonal growth to accelerate the end-to-end connection. Currently, there are no conduits equipped with both electrical conduction and axon-guiding surface structure. Herein, a biodegradable, conductive poly(l-lactide-co-caprolactone)/graphene (PLCL/GN) composite conduit is designed. The conduit with 20.96 ± 1.26 MPa tensile strength has a micropatterned surface of 20 µm groove fabricated by microimprint technology and self-assembled polydopamine (PDA). In vitro evaluation shows that the conduits with ES effectively stimulate the directional cell migration, adhesion, and elongation, and enhance neuronal expression of SCs. The rat sciatic nerve crush model demonstrates that the conductive micropatterned conduit with ES promotes the growth of myelin sheath, faster nerve regeneration, and 20-fold functional recovery in vivo. These discoveries prove that the PLCL(G)/PDA/GN composite conduit is a promising tool for PNI treatment by providing the functional integration of physical guidance, biomimetic biological regulation, and bioelectrical stimulation, which inspires a novel therapeutic approach for nerve regeneration in the future.

Paradoxical Changes of Cutaneous Microcirculation and Sympathetic Fibers of Rat Hind Limbs after Sciatic Nerve Compression.

BACKGROUND: Recent studies show evidence that surgical nerve decompression could improve cutaneous blood flow (CBF), which might benefit ulcer healing. However, the change of CBF and sympathetic fibers after nerve compression is poorly understood. In the current study, a unilateral sciatic nerve compression model was created in Sprague-Dawley rats. METHODS: A laser Doppler imaging system was applied to assess the CBF of the regions below the ankles. Immunohistochemistry and transmission electron microscopy were used to investigate the histopathologic changes of sympathetic fibers in sciatic nerve samples. RESULTS: Laser Doppler imaging revealed decreased CBF of both the lesional limb and the contralesional limb, which occurred earlier in the lesional side, indicating an enhanced sympathetic tone on vasomotor function. Intraneural density of sympathetic fibers decreased on both sides and the ultrastructure of unmyelinated fibers of both sides degenerated in a nonsynchronized manner. CONCLUSIONS: The study revealed nonsynchronized reduced CBF of bilateral hind limbs with paradoxically degenerated and diminished sympathetic fibers in bilateral sciatic nerves after unilateral sciatic nerve compression. These results may validate the importance of and broaden the indications for surgical nerve decompression in preventing or treating foot ulcers.

Injectable nanofiber-reinforced bone cement with controlled biodegradability for minimally-invasive bone regeneration.

Injectable materials show their special merits in regeneration of damaged/degenerated bones in minimally-invasive approach. Injectable calcium phosphate bone cement (CPC) has attracted broad attention for its bioactivity, as compared to non-degradable polymethyl methacrylate cement. However, its brittleness, poor anti-washout property and uncontrollable biodegradability are the main challenges to limit its further clinical application mainly because of its stone-like dense structure and fragile inorganic-salt weakness. Herein, we developed a kind of injectable CPC bone cement with porous structure and improved robustness by incorporating poly(lactide-co-glycolic acid) (PLGA) nanofiber into CPC, with carboxymethyl cellulose (CMC) to offer good injectability as well as anti-wash-out capacity. Furthermore, the introduction of PLGA and CMC also enabled a formation of initial porous structure in the cements, where PLGA nanofiber endowed the cement with a dynamically controllable biodegradability which provided room for cell movement and bone ingrowth. Interestingly, the reinforced biodegradable cement afforded a sustainable provision of Ca2+ bioactive components, together with its porous structure, to improve synergistically new bone formation and osteo-integration in vivo by using a rat model of femur condyle defect. Further study on regenerative mechanisms indicated that the good minimally-invasive bone regeneration may come from the synergistic enhanced osteogenic effect of calcium ion enrichment and the improved revascularization capacity contributed from the porosity as well as the lactic acid released from PLGA nanofiber. These results indicate the injectable bone cement with high strength, anti-washout property and controllable biodegradability is a promising candidate for bone regeneration in a minimally-invasive approach.

Celecoxib activates autophagy by inhibiting the mTOR signaling pathway and prevents apoptosis in nucleus pulposus cells.

BACKGROUND: Intervertebral disc degeneration results from a variety of etiologies, including inflammation and aging. Degenerated intervertebral discs feature down-regulated extracellular matrix synthesis, resulting in losing their ability to retain water and absorb compression. Celecoxib is a well-known selective cyclooxygenase-2 inhibitor for treating arthritis and relieving pain. Nevertheless, the mechanism of Celecoxib for treating inflammation-related intervertebral disc degeneration has not yet been clarified. METHOD: Protein synthesis was analyzed by western blot. Fluorescent probes DCFH-DA and MitoSox Red detected reactive oxygen species and were measured by flow cytometry. The activity of the kinase pathway was evaluated by protein phosphorylation. Autophagy was monitored by mRFP-GFP-LC3 transfection and LC3 analysis. Mitochondrial apoptotic proteins were analyzed by western blot and cell membrane integrity was measured by flow cytometry. The autophagic gene was silenced by siRNA. RESULTS: In this study, interleukin-1ß stimulation reduced the synthesis of aggrecan, type I and II collagen and caused excessive production of reactive oxygen species. We looked for a therapeutic window of Celecoxib for nucleus pulposus cells to regain extracellular matrix synthesis and reduce oxidative stress. To look into nucleus pulposus cells in response to stimuli, enhancement of autophagy was achieved by Celecoxib, confirmed by mRFP-GFP-LC3 transfection and LC3 analysis. The mammalian target of rapamycin and a panel of downstream proteins responded to Celecoxib and propelled autophagy machinery to stabilize homeostasis. Ultimately, inhibition of autophagy by silencing autophagy protein 5 disrupted the protective effects of Celecoxib, culminating in apoptosis. CONCLUSION: In summary, we have demonstrated a new use for the old drug Celecoxib that treats intervertebral disc degeneration by enhancing autophagy in nucleus pulposus cells and opening a door for treating other degenerative diseases.

Siglec15 Checkpoint Blockade for Simultaneous Immunochemotherapy and Osteolysis Inhibition in Lung Adenocarcinoma Spinal Metastasis via a Hollow Nanoplatform.

Low responsiveness to anti-programmed death-1/programmed death-ligand 1 (anti-PD-1/PD-L1) for solid tumors indicates the presence of other immunosuppressive pathways. Siglec15, a newly discovered immune checkpoint, has been reported to repress immune responses in the tumor microenvironment (TME) and regulate osteoclast differentiation. However, the role of Siglec15 in the treatment for bone metastasis remains unclear. Herein, Siglec15 shows significantly higher expression in lung adenocarcinoma spinal metastasis (LUAD-SM) than in para-cancerous spinal tissues and primary LUAD. Subsequently, a TME-responsive hollow MnO2 nanoplatform (H-M) loaded with Siglec15 siRNA and cisplatin (H-M@siS15/Cis) is developed, and the surface is modified with an aspartic acid octapeptide (Asp8 ), thus allowing H-M to target spinal metastasis. High drug-loading capacity, good biocompatibility, effective tumor accumulation, and efficient Siglec15 silencing are demonstrated. Furthermore, the nanoparticles could reverse immunosuppression caused by tumor cells and tumor-associated macrophages (TAMs) and inhibit osteoclast differentiation via Siglec15 downregulation in vitro. In a LUAD-SM mouse model, H-M@siS15/Cis-Asp8 exhibits superior therapeutic efficacy via synergetic immunochemotherapy and osteolysis inhibition. Taken together, this single nanoplatform reveals the therapeutic potential of the new immune checkpoint Siglec15 in LUAD-SM and provides a strategy to treat this disease.

Siglec15 facilitates the progression of non-small cell lung cancer and is correlated with spinal metastasis.

Background: Non-small cell lung cancer (NSCLC) frequently metastasizes to bone, leading to poor prognosis. Siglec15 has been identified as a newly discovered immune checkpoint and exists in a variety of tumors. However, the expression and function of Siglec15 in NSCLC and bone metastasis remains largely unclear. Methods: Siglec15 expression in NSCLC and the correlation between Siglec15 expression and the clinicopathological factors of patients with NSCLC were analyzed using The Cancer Genome Atlas (TCGA) dataset. Correlation analysis between Siglec15 and bone metastasis-related genes expression was based on the Molecular Signatures Database (MSigDB). Western blotting and immunohistochemistry were applied to detect Siglec15 expression in NSCLC and spinal metastasis. Human A549 and mouse CMT167 cells were transfected with Siglec15 siRNA to investigate its biological functions in NSCLC proliferation, migration, and invasion. The immune-related signaling pathways and correlations between Siglec15 and tumor-infiltrating immune cells and different immune checkpoints in the NSCLC tumor microenvironment (TME) were analyzed using Estimating Relative Subsets of RNA Transcripts (CIBERSORT) and gene set enrichment analysis (GSEA). To demonstrate Siglec15 in NSCLC cell-mediated T cell suppression and investigate the potential mechanism of Siglec15 silencing in antitumor immunity, we used a T cell killing assay in vitro and the high­throughput sequencing approach. Results: Siglec15 expression was positively associated with the tumor stage and lymph node metastasis, and was markedly up-regulated in NSCLC bone metastasis. Functionally, Siglec15 knockdown inhibited the proliferation, migration, and invasion of NSCLC cells (A549 and CMT167 cell lines). A total of eight kinds of tumor-infiltrating immune cells were found to have a strong association with the Siglec15 expression in NSCLC cases. The expression of previously discovered immune checkpoints was higher in the high Siglec15 expression NSCLC group. Furthermore, an in vitro T cell killing assay showed that the down-regulation of Siglec15 in tumor cells could enhance the antitumor immune responses of CD8+ T cells. High­throughput sequencing revealed the potential molecular mechanisms underlying the Siglec15-mediated immunosuppression effect of tumor cells on immune cells. Conclusions: Siglec15 may be involved in the pathogenesis of spinal metastasis in NSCLC and provide a new potential therapeutic target for the treatment of NSCLC and bone metastasis.

Effect of different structures fabricated by additive manufacturing on bone ingrowth.

OBJECTIVE: To study the effects of different structures (solid/hollow) and pore diameters (300/600 µm) on bone ingrowth. METHODS: Porous titanium alloy scaffolds (3.2 * 10.5 mm) were printed using electron beam melting. The implants were divided into either Hollow or Solid Group. The upper half of each implant was printed with a pore diameter of 600 µm while the bottom half was printed with a pore diameter of 300 µm. Visualization of the structural morphology was done using Scanning Electron Microscope (SEM). Cell proliferation was evaluated with the cell counting kit-8 assay and live/dead staining assay. The different lateral femoral condyles of 15 New Zealand rabbits were implanted with different groups of scaffolds. The rabbits were randomly sacrificed at the 4th, 8th, and 12th week postoperatively. Bone mineral density (BMD) and bone volume fraction (BV/TV) evaluation was completed by quantitative Micro-Computed Tomography (Micro-CT). Tissue histology were stained with toluidine blue to observe bone ingrowth under an optical microscope, and the percentage of new bone area were calculated using Image Pro-Plus 6.0. RESULTS: SEM images showed a significant decrease in residual powder in the hollow implant and cell studies showed no obvious cytotoxicity for the Ti6Al4V scaffolds. Micro-CT reconstruction revealed high levels of new bone formation around the scaffolds. The trabeculae around the implants showed a gradual increase with each week, and new bone filled the scaffold pores gradually. BMD, BV/TV, and tissue histology revealed the 300 µm pore diameter is more conducive to bone ingrowth than the 600 µm (p < .05). CONCLUSION: Our study revealed that Ti6Al4V implants with hollow structure could reduce the residual metal powder and implants with 300 µm pore diameter were more effective on bone formation than a 600 µm.

Oroxin B Attenuates Ovariectomy-Induced Bone Loss by Suppressing Osteoclast Formation and Activity.

BACKGROUND: Osteoclasts are the major players in bone resorption and have always been studied in the prevention and treatment of osteoporosis. Previous studies have confirmed that a variety of flavonoids inhibit osteoporosis and improve bone health mainly through inhibiting osteoclastogenesis. Oroxin B (OB) is a flavonoid compound extracted from traditional Chinese herbal medicine Oroxylum indicum (L.) Vent, exerts potent antitumor and anti-inflammation effect, but its effect on osteoclastogensis remains unknown. METHODS: We comprehensively evaluated the effect of OB on the formation and function of osteoclasts and the underling mechanism by bone marrow-derived macrophage in vitro. In vivo, we used mice ovariectomized model to verify the protective effect of OB. RESULTS: OB was found to inhibit osteoclast formation and bone resorption function in vitro, in a dose-dependent manner and the increased osteoclastic-related genes induced by RANKL (NFATc1, c-fos, cathepsin K, RANK, MMP9 and TRAP) were also attenuated following OB treatment. Mechanistical investigation showed OB abrogated the increased phosphorylation level of MAPK and NF-κB pathway, and diminished the expression of the vital transcription factors for osteoclastogenesis. OB also prevented ovariectomy (OVX)-induced bone loss by inhibiting osteoclast formation and activity in mice. CONCLUSION: Our study demonstrated that OB may act as an anti-osteoporosis agent by inhibiting osteoclast maturation and attenuating bone resorption.

Hypoxia Inducible Factor-1α Is a Regulator of Autophagy in Osteoarthritic Chondrocytes.

OBJECTIVE: To investigate the relationship between hypoxia inducible factor-1α (HIF-1α) and the autophagic response in osteoarthritic chondrocytes (OA), under inflammatory insult as represented by in vitro OA model. METHODS: Human chondrocyte cell line C28/I2 was cultured in both normoxic and hypoxic conditions and treated with interleukin-1ß (IL1ß) to emulate OA inflammatory insult in vitro. Cellular HIF-1α expression was silenced using siRNA transfection and cellular autophagic (P62/LC3II) response and OA chondrocyte damage (COL2A1/MMP13) related proteins were examined using western blotting. Cellular mitophagic (BNIP3/PINK1/Parkin) and apoptotic (Caspase/Cleaved Caspase 3) were also evaluated to assess mitophagy-mediated cell death due to HIF-1α silencing. RESULTS: Chondrocyte basal autophagy levels were higher in a HIF-1α elevated environment and was more resistant to IL1ß-induced inflammatory insult. Increase in autophagic proteins showed better chondrocyte repair, which resulted a lower level of reactive oxygen species production, and lesser damage to chondrocyte integrity. Silencing HIF-1α activates cellular PINK1/Parkin and BNIP3 mitophagic proteins, which leads to the activation of Caspase/Cleaved Caspase 3 apoptotic cascade. CONCLUSION: Our results show that chondrocyte autophagy is dependent on HIF-1α expression, showing the importance of HIF-1α in hypoxic chondrocyte function in OA. Dysregulation of HIF-1α expression results in the activation of mitophagy-mediated apoptosis.

Clinical outcomes of lumbar spinal surgery in patients 80 years or older with lumbar stenosis or spondylolisthesis: a systematic review and meta-analysis.

PURPOSE: This systematic review and meta-analysis of all available evidence was performed to assess the safety and efficacy of surgery for lumbar stenosis and spondylolisthesis in patients 80 years or older versus those younger than 80 years. METHODS: A search of the literature was conducted in PubMed/MEDLINE, EMBASE and the Cochrane Collaboration Library. Relevant studies comparing the clinical outcomes of lumbar surgery in octogenarians and younger patients were selected according to the eligibility criteria. The predefined endpoints were extracted and meta-analysed from the identified studies. RESULTS: Data from 16 observational studies including 374,197 patients were included in the final analysis. The pooled data revealed that patients 80 years or older had a significantly higher incidence of overall complication, mortality, readmission and longer length of hospital stay than younger patients. There was a similar improvement in the clinical symptoms (Oswestry Disability Index and pain) of patients in the two groups. No significant differences in overall wound complication, reoperation rate, operative time and intraoperative blood loss were found between the groups. CONCLUSIONS: Our results revealed that the clinical improvement in pain and disability did not significantly differ according to age, although the patients aged 80 years or older had increased incidences of mortality and complication than younger patients. Age alone is not a contraindication for lumbar surgery in very old patients. A careful preoperative evaluation, proper patient selection and appropriate surgical approach are important to achieve successful surgical outcomes. These slides can be retrieved under Electronic Supplementary Material.

Epidemiology of ossification of the spinal ligaments and associated factors in the Chinese population: a cross-sectional study of 2000 consecutive individuals.

BACKGROUND: The epidemiology and cause of ossification of the spinal ligaments (OSL) remains obscure. To date, there is no study that comprehensively evaluates the prevalence, distribution, and concomitance of each type of OSL by CT among general Chinese population. We therefore aimed to comprehensively investigate epidemiological characteristics of OSL using whole spine CT in the Chinese population and examine the factors that correlate with the presence of OSL. METHODS: Ossification of the posterior longitudinal ligament (OPLL), ligamentum flavum (OLF), anterior longitudinal ligament (OALL), nuchal ligament (ONL), and diffuse idiopathic skeletal hyperostosis (DISH) were evaluated from the subjects who underwent PET/CT for the purpose of cancer screening in our hospital. Prevalence, distribution, and concomitance of OSL were reviewed. Logistic regression analysis was performed to identify the risk factors of OSL. RESULTS: A total of 2000 subjects (1335 men and 665 women) were included. The prevalence rate of cervical OPLL (C-OPLL) was 4.1%, thoracic OPLL (T-OPLL) 2.25%, lumbar OPLL (L-OPLL) 0.8%, thoracic OLF (T-OLF) 37.65%, lumbar OLF (L-OLF) 1.45%, ONL 31.5%, DISH 3.85%. The most commonly involved level was C5 for C-OPLL, T1 for T-OPLL, T10 for T-OLF, and T8/9 for OALL. 21% of subjects with C-OPLL had T-OPLL, 44% of C-OPLL had T-OLF, 38% of T-OPLL had C-OPLL, 53% of T-OPLL had T-OLF, 44% of L-OPLL had T-OPLL, and 56% of L-OPLL had T-OLF. The average age of OSL-positive subjects was significantly higher than that of OSL-negative subjects. The results of the multiple regression analysis revealed that males had a strong association with DISH (odds ratio, 3.15; 95% confidence interval, 1.27-7.78; P = 0.013). CONCLUSION: The prevalence of OSL in the Chinese was revealed. Tandem ossification is not uncommon in people with OSL. There is a high incidence of multiple-regional OPLL in the whole spine. Approximately half of the subjects with OPLL coexist with T-OLF. For patients with clinical symptoms induced by OPLL, thorough evaluation of whole spine using CT is recommended.