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Discectomy and interbody fusion are widely used in the treatment of intervertebral disc-related diseases. Among them, the interbody cage plays a significant role. However, the complications related to the interbody cage, such as nonunion or pseudoarthrosis, subsidence, loosening, and prolapse of the cage, cannot be ignored. By changing the design and material of the interbody fusion cage, a better fusion effect can be obtained, the incidence of appeal complications can be reduced, and the quality of life of patients after interbody fusion can be improved.This study reviewed the research progress of cage design and material and discussed the methods of cage design and material to promote intervertebral fusion.Current treatment of cervical and lumbar degenerative disease requires interbody fusion to maintain decompression and to promote fusion and reduce the incidence of fusion failure through improvements in implant material, design, internal structure, and function. However, interbody fusion is not an optimal solution for treating vertebral instability.

Background Limited progress in terms of an effective treatment for spinal cord injury (SCI) emphasizes the urgent need for novel therapies. As a vital central nervous system component, the resident astrocytes play crucial roles in regulating recovery after SCI. In this study, recovery after SCI was compared following the transplantation of either A1 or A2 astrocytes. A1 astrocytes are harmful as they upregulate the neurotoxic classical complement cascade genes. Conversely, A2 astrocytes are characterized as neuroprotective as they upregulate the production of many neurotrophic factors. Methods We used different supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4 to generate A1 and A2 astrocytes. We detected the influence of astrocytes on neurons by co-culturing A1 and A2 astrocytes with neurons. We transplanted astrocytes into the lesion site of the spinal cord and assessed lesion progression, neural restoration, glia formation and locomotor recovery. Results Astrocytes were polarized into A1 and A2 phenotypes following culture in the supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4, respectively. Furthermore, co-culturing A2 astrocytes with neurons significantly suppressed glutamate-induced neuronal apoptosis and promoted the degree of neuron arborization. Transplantation of these A2 astrocytes into the lesion site of the spinal cord of mice significantly improved motor function recovery, preserved spared supraspinal pathways, decreased glia scar deposition, and increased neurofilament formation at the site of injury compared to the transplantation of A1 astrocytes. Additionally, enhanced A2 astrocytes with potentially beneficial A2-like genes were also detected in the A2 group. Moreover, luxol fast blue staining and electron microscopy indicated increased preservation of myelin with organized structure after transplantation of A2 astrocytes than of A1 astrocytes. Conclusions A2 astrocyte transplantation could be a promising potential therapy for SCI. Graphical Abstract

The role of CD93 in inflammatory response has been reported in multiple previous studies. However, the underlying mechanism of CD93 in microglial activation and migration during neuroinflammation post spinal cord injury (SCI) remains elusive. In the current study, we performed western blot, qRT-PCR, immunofluorescence analyses Transwell assay, and ELISA to determine the expression change and in-depth molecular mechanism of CD93 in microglia post inflammatory initiation. We found that CD93 expression was increased in microglia after SCI in vivo or lipopolysaccharide (LPS) stimuli in vitro. Additionally, CD93 interacted with TAK1 to inhibit NF-κB activation, thus attenuating inflammation and migration of microglia after treatment with LPS. These findings indicate that CD93 might participate in microglia-induced neuroinflammation development post SCI, suggesting that CD93 is a promising target for neuroimmunological regulation.

BackgroundNeuroinflammation following spinal cord injury (SCI) results in prolonged neurological damage and locomotor dysfunction. Polarization of microglia is vital to regulation of neuroinflammation, although the underlying mechanisms have not yet been elucidated. Endocannabinoid receptor subtype 2 (CB2R) is reported to ameliorate neurodegeneration via immunomodulation activities. However, the underlying machinery in the context of SCI remains unclear.MethodsA lipopolysaccharide-induced microglia inflammation model and a mouse model of SCI were employed to investigate the regulatory role of CB2R in the polarization of microglia in response to excess neuroinflammation. Markers of inflammation and autophagy were measured by Western blot analysis, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. Histological staining with hematoxylin and eosin, Nissl, and Luxol® fast blue was conducted using commercial kits. The locomotor function of the hindlimbs of the experimental mice was evaluated with the Basso Mouse Scale, Louisville Swim Scale, and footprint assay.ResultsThe results showed that CB2R promoted M2 differentiation, increased interleukin (IL)-10 expression, and inhibited M1 differentiation with decreased expression of IL-1β and IL-6. CB2R activation also increased ubiquitination of the NLRP3 inflammasome and interacted with the autophagy-related proteins p62 and microtubule-associated proteins 1B light chain 3. Treatment with the CB2R activator JWH-133 reduced loss of myelin, apoptosis of neurons, and glial scarring, leading to improved functional recovery of the hindlimbs, while the CB2R antagonist AM630 produced opposite results.ConclusionTaken together, these results suggested that CB2R activation attenuated neuroinflammation targeting microglial polarization by promoting NLRP3 clearance, thereby facilitating functional recovery post-SCI.

Spinal cord injury (SCI) is a destructive polyneuropathy that can result in loss of sensorimotor function and sphincter dysfunction, and even death in critical situations. MicroRNAs (miRs) are a series of non-coding RNA molecules that are involved in transcriptional regulation. Previous studies have demonstrated that modulation of multiple miRs is involved in neurological recovery after SCI. However, the functions of miR-340-5p in SCI remain uncertain. Therefore, we probed the therapeutic effect and mechanism of miR-340-5p in microglia in vitro and in vivo in SCI rats. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were employed to examine the alterations in miR-340-5p and P38 levels in SCI rats. miR-340-5p targets in microglia were ascertained using luciferase reporter assays, immunofluorescence analyses, and western blotting. We also established an SCI model and administered miR-340-5p. The effects of miR-340-5p on the amelioration of inflammation, oxidative stress, and apoptosis following SCI were assessed using immunofluorescence, immunohistochemistry, and histological analyses. Finally, locomotor function recovery was determined using the Basso, Beattie, Bresnahan rating scale. In our study, the expression profiles and luciferase assay results clarified that P38 was a target of miR-340-5p, which was associated with activation of the P38-MAPK signaling pathway. Elevation of miR-340-5p decreased P38 expression, subsequently inhibiting the inflammatory reaction. SCI-induced secondary neuroinflammation was relieved under miR-340-5p treatment. Moreover, by controlling neuroinflammation, the increased levels of miR-340-5p might counter oxidative stress and reduce the degree of apoptosis. We also observed decreasing gliosis and glial scar formation and increasing neurotrophin expression at the chronic stage of SCI. Together, these potential effects of miR-340-5p treatment ultimately improved locomotor function recovery in SCI rats.

BackgroundSpinal cord injury (SCI) is a severe traumatic disorder of the central nervous system (CNS) that causes irreversible damage to the nervous tissue. The consequent hemorrhage contributed by trauma induces neuronal ferroptosis post SCI, which is an important death mode to mediate neuronal loss. Growth differentiation factor 15 (GDF15) is a cytokine that regulates cell proliferation, differentiation, and death. However, the specific role of GDF15 in neuronal ferroptosis post SCI remains unknown.Materials and MethodsNeuronal ferroptosis in vitro was measured by detection of lipid peroxidation, glutathione, iron content, and reactive oxidative stress. In vivo, western blotting and immunofluorescence (IF) staining was utilized to measure ferroptosis post SCI. IF staining, TUNEL staining, hematoxylin-eosin staining, and Nissl staining were used to measure neurological damage. Finally, locomotor function recovery was analyzed using the Basso Mouse Scale and Louisville Swim Scale.ResultsGDF15 was significantly increased in neuronal ferroptosis and silencing GDF15 aggravated ferroptosis both in vitro and in vivo. Besides, GDF15-mediated inhibition of neuronal ferroptosis is through p62-dependent Keap1-Nrf2 pathway. In SCI mice, knockdown of GDF15 significantly exacerbated neuronal death, interfered with axon regeneration and remyelination, aggravated ferroptosis-mediated neuroinflammation, and restrained locomotor recovery.ConclusionGDF15 effectively alleviated neuronal ferroptosis post SCI via the p62-Keap1-Nrf2 signaling pathway and promoted locomotor recovery of SCI mice, which is suggested as a potential target on SCI pathogenesis and treatment.

Background Traumatic spinal cord injury (SCI)-induced neuroinflammation results in secondary neurological destruction and functional disorder. Previous findings showed that microglial pyroptosis plays a crucial role in neuroinflammation. Thus, it is necessary to conduct a comprehensive investigation of the mechanisms associated with post-SCI microglial pyroptosis. The Fanconi Anemia Group C complementation group gene (FANCC) has been previously reported to have an anti-inflammation effect; however, whether it can regulate microglial pyroptosis remains unknown. Therefore, we probed the mechanism associated with FANCC-mediated microglial pyroptosis and neuroinflammation in vitro and in vivo in SCI mice. Methods Microglial pyroptosis was assessed by western blotting (WB) and immunofluorescence (IF), whereas microglial-induced neuroinflammation was evaluated by WB, Enzyme-linked immunosorbent assays and IF. Besides, flow cytometry, TdT-mediated dUTP Nick-End Labeling staining and WB were employed to examine the level of neuronal apoptosis. Morphological changes in neurons were assessed by hematoxylin–eosin and Luxol Fast Blue staining. Finally, locomotor function rehabilitation was analyzed using the Basso Mouse Scale and Louisville Swim Scale. Results Overexpression of FANCC suppressed microglial pyroptosis via inhibiting p38/NLRP3 expression, which in turn reduced neuronal apoptosis. By contrast, knockdown of FANCC increased the degree of neuronal apoptosis by aggravating microglial pyroptosis. Besides, increased glial scar formation, severe myelin sheath destruction and poor axon outgrowth were observed in the mice transfected with short hairpin RNA of FANCC post SCI, which caused reduced locomotor function recovery. Conclusions Taken together, a previously unknown role of FANCC was identified in SCI, where its deficiency led to microglia pyroptosis, neuronal apoptosis and neurological damage. Mechanistically, FANCC mediated microglia pyroptosis and the inflammatory response via regulating the p38/NLRP3 pathway.

Spinal cord injury (SCI) is a ruinous neurological pathology that results in locomotor and sensory impairment. Neuro-inflammation and secondary neuronal apoptosis contribute to SCI, with anti-inflammatory therapies the focus of many SCI studies. Forsythoside B (FTS•B), a phenylethanoid glycoside extracted from the leaves of Lamiophlomis rotata Kudo, has been shown previously to have anti-inflammatory properties. Nevertheless, the therapeutic effect of FTS•B on neuro-inflammation after SCI is unknown.Neuro-inflammation was assessed by western blotting (WB), immunofluorescence (IF) staining, and enzyme-linked immunosorbent assay (ELISA) both in vitro and in vivo. Secondary neuronal apoptosis was simulated in a microglia-neuron co-culture model with the degree of apoptosis measured by WB, IF, and TUNEL staining. In vivo, FTS•B (10 mg/kg, 40 mg/kg) were intraperitoneally injected into SCI mice. Morphological changes following SCI were evaluated by Nissl, Hematoxylin-eosin, and Luxol Fast Blue staining. Basso Mouse Scale scores were used to evaluate locomotor function recovery.FTS•B markedly decreased the levels of iNOS, COX-2 and signature mediators of inflammation. Phosphorylated p38 and nuclear factor-kappa B (NF-κB) were markedly decreased by FTS•B. Additionally, FTS•B-induced inhibition of NF-κB and p38-MAPK signaling pathways was reversed by Nrf2 downregulation. Administration of FTS•B also significantly reduced apoptosis-related protein levels indicating that FTS•B ameliorated secondary neuronal apoptosis. FTS•B administration inhibited glial scar formation, decreased neuronal death, tissue deficiency, alleviated demyelination, and promoted locomotor recovery.FTS•B effectively attenuates neuro-inflammation and secondary neuronal apoptosis by inhibition of NF-κB and p38-MAPK signaling pathways through activating Nrf2 after SCI. This study demonstrates FTS•B to be a potential therapeutic for SCI.

Excessive reactive oxygen species (ROS) and apoptosis in nucleus pulposus (NP) cells accelerate the process of intervertebral disc degeneration (IDD). Here, we integrated pathological samples and in vitro and in vivo framework to investigate the impact of phosphorylation of eukaryotic initiation factor-2α (eIF2α)/activating transcription factor 4 (ATF4)/Indian hedgehog (Ihh) signaling in the IDD. From the specimen analysis of the IDD patients, we found phosphorylated eIF2α (p-eIF2α), ATF4 and Ihh protein levels were positively related while the NP tissue went degenerative. In vitro, tumor necrosis factor (TNF)-α caused the NP cell degeneration and induced a cascade of upregulation of p-eIF2α, ATF4, and Ihh. Interestingly, ATF4 could enhance Ihh expression through binding its promoter region, and silencing of ATF4 decreased Ihh and protected the NP cells from degeneration. Moreover, ISRIB inhibited the p-eIF2α, which resulted in a suppression of ATF4/Ihh, and alleviated the TNF-α-induced ROS production and apoptosis of NP cells. On the contrary, further activating p-eIF2α aggravated the NP cell degeneration, with amplification of ATF4/Ihh and a higher level of ROS and apoptosis. Additionally, applying cyclopamine (CPE) to suppress Ihh was efficient to prevent NP cell apoptosis but did not decrease the ROS level. In an instability-induced IDD model in mice, ISRIB suppressed p-eIF2α/ATF4/Ihh and prevented IDD via protecting the anti-oxidative enzymes and decreased the NP cell apoptosis. CPE prevented NP cell apoptosis but did not affect anti-oxidative enzyme expression. Taken together, p-eIF2α/ATF4/Ihh signaling involves the ROS level and apoptosis in NP cells, the pharmacological disruption of which may provide promising methods in preventing IDD.

Background Emerging evidence has indicated that abnormal microRNAs (miRNAs) play critical roles in carcinogenesis and progression of osteosarcoma (OS). The aim of this study was to clarify the relationship between miR-766-3p expression and osteosarcoma development and to explore its potential mechanism. Methods miR-766-3p was the most downregulated miRNA by analyzing GSE65071 from the GEO database. RT-PCR and western blot was performed to determine miR-766-3p expression and its specific target gene in human OS samples and cell lines. CCK-8 proliferation, colony formation, EdU, wound-healing, and transwell assays were used respectively to evaluate the influences of miR-766-3p depletion or ectopic expression on OS proliferation, migration and invasion in vitro. And a mouse tumorigenicity model was conducted to investigate effects of miR-766-3p in vivo. Moreover, we identified directly interactions between miR-766-3p and its specific target gene using luciferase reporter assays. Results miR-766-3p expression was overexpressed in OS tissues and cell lines, and ectopic miR-766-3p expression repressed the malignant level of OS, including cell proliferation, migration, invasion and epithelial to mesenchymal transition (EMT) in vitro and in vivo. B-Cell Lymphoma 9-Like Protein (BCL9L) was negatively correlated with miR-766-3p expression in human OS tissue, and was validated as a downstream target of miR-766-3p by the luciferase reporter assay and Western blotting. Rescue experiment indicated that BCL9L could restore the effects of miR-766-3p on OS migration and invasion. The β-Catenin signaling pathway was demonstrated as being implicated in the miR-766-3p/BCL9L axis. Conclusions In conclusion, miR-766-3p is a negative regulator of BCL9L and a risk factor for tumor metastasis in OS progression.

Osteosarcoma (OS) is one of the most common primary bone malignancies and accounts for 3.4% of pediatric tumors. Its 5-year survival is as low as about 20%. Differentially expressed lncRNAs in OS profiling were searched in the downloaded profile of GSE12865. As a result, LINC00266-1 was detected to be upregulated in both GSE12865 and OS tissues we collected. SMAD2 was the downstream target binding to promoter sites of LINC00266-1, displaying a positive regulatory interaction. Knockdown of LINC00266-1 suppressed the proliferative and metastatic abilities, and promoted the apoptosis in OS cells. Besides, knockdown of LINC00266-1 significantly alleviated the growth of OS in vivo. MiR-548c-3p was the sponge miRNA of LINC00266-1, which was able to reverse the regulatory effects of LINC00266-1 on OS cell phenotypes. Moreover, miR-548c-3p bound to the 3′-UTR of SMAD2 and thus downregulated SMAD2. Overexpression of SMAD2 partially reversed the regulatory effects of LINC00266-1 on OS cell phenotypes. Finally, we have identified that LINC00266-1/miR-548c-3p/SMAD2 feedback loop was responsible for stimulating the development of OS.

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Background: To uncover the role of LINC00266-1 in the development of osteosarcoma (OS) and identify the underlying mechanism.Methods: LINC00266-1 levels in OS tissues in the GSE12865 dataset were analyzed. Binding sites between the DNA of LINC00266-1 and SMAD2 and potential miRNAs binding LINC00266-1 were predicted via databases. The effects of LINC00266-1 on the proliferative, migratory, invasive and apoptotic abilities were evaluated by a series of functional experiments. Interactions in the LINC00266-1/miR-548c-3p/SMAD2 feedback loop were assessed by RNA immunoprecipitation (RIP), Chromatin immunoprecipitation (ChIP)-qPCR and dual luciferase reporter assays. Tumorigenesis assay was then conducted in nude mice. The potential influence of LINC00266-1 on the positive expression of Ki67 in nude mice bearing OS xenografts was elucidated through immunohistochemistry.Results: LINC00266-1 was upregulated in OS tissues and cells. SMAD2 was able to bind sites in the LINC00266-1 promoter and thus upregulate LINC00266-1. Knockdown of LINC00266-1 suppressed the proliferative and metastatic abilities but promoted the apoptosis of OS cells. LINC00266-1 sponged miR-548c-3p, an action that partially reversed the regulatory effects of LINC00266-1 on OS cell phenotypes. Moreover, miR-548c-3p bound to the 3’-UTR of SMAD2 and thus downregulated SMAD2. Overexpression of SMAD2 partially reversed the regulatory effects of LINC00266-1 on OS cell phenotypes. Knockdown of LINC00266-1 significantly alleviated the growth of OS in vivo.Conclusions: The LINC00266-1/miR-548c-3p/SMAD2 feedback loop stimulates the development of OS.

Neuroinflammation following spinal cord injury (SCI) leads to extensive secondary damage in neural tissue adjacent to the primary lesion foci. 5-Methoxytryptophan (5MTP) is a metabolite of tryptophan and proven to play a protective role in several inflammation-related diseases. However, the specific efficacy and molecular mechanism of 5MTP in SCI remains unknown. Here, we aimed to investigate the anti-inflammatory role of 5MTP in microglia-induced neuroinflammation and its therapeutic effect in SCI. To assess the effect of 5MTP in neuroinflammation, we used lipopolysaccharide (LPS) to stimulate microglia in vitro and detected the microglial phenotype using immunofluorescence staining, the inflammatory-related pathway using western blotting, and pro-inflammatory cytokines using ELISA and immunofluorescence. To explore the therapeutic effect of 5MTP in SCI, we performed contusion of the spinal cord in mice and measured the levels of neuroinflammation, glial accumulation, histological and functional recovery using ELISA, immunofluorescence staining, immunohistochemical staining, hematoxylin-eosin staining, Nissl staining and the Basso Mouse Scale, respectively. We found that treatment with 5MTP contributed to decreased activation of pro-inflammatory microglia and reduced the generation of inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IL-18, by negative regulation of the p38-MAPK signaling pathway and NLRP3/caspase-1 expression. In vivo, administration of 5MTP showed mitigatory neuroinflammation levels associated with alleviated glial scar in SCI mice; hence, the neurological integrity and the neuronal survival, as well as locomotor function, were improved following 5MTP administration. 5MTP, as a novel anti-neuroinflammatory reagent, can attenuate activated microglia-induced secondary injury following SCI, and therefore, shows promise as a potential compound for application in a clinical trial for SCI therapy.

We aimed to investigate the biological functions of circLRP6 in the progression of osteosarcoma. CircLRP6 level in OS was detected by quantitative real-time polymerase chain reaction. Correlation between circLRP6 level with survival of OS patients was evaluated. Cell counting kit-8 and Transwell assay were conducted to detect proliferative, migratory and invasive capacities of OS cells. Cell cycle and apoptosis in OS cells influenced by circLRP6 were evaluated by flow cytometry. RNA immunoprecipitation was conducted to verify the binding relationship between circLRP6 with LSD1 and EZH2. Finally, the interaction between LSD1, EZH2 and promoter regions of KLF2, APC was clarified by chromatin immunoprecipitation. CircLRP6 level markedly increased in OS tissues. Besides, OS patients with high expression of circLRP6 showed shorter disease-free survival and over-all survival than those with low expression. CircLRP6 knockdown suppressed proliferative, migratory and invasive rates of OS cells. Moreover, circLRP6 knockdown induced apoptosis and arrested cell cycle in G0/G1 phase. The interaction between circLRP6 with LSD1 and EZH2 mediates their binding to the promoter regions of KLF2 and APC. Knockdown of circLRP6 weakened the binding abilities of LSD1, EZH2 to KLF2, APC. APC overexpression inhibited proliferation, induced apoptosis and arrested cell cycle. Moreover, the tumor-suppressor effect of downregulated circLRP6 on OS could be reversed by APC knockdown. Collectively, circLRP6 was highly expressed in OS and served as an oncogene by binding to LSD1 and EZH2 to inhibit expressions of KLF2 and APC.