Improving Hybrid Tin Halide Films by Tin Trifluoromethanesulfonate for Lead-Free Perovskite Solar Cells.

Tin-based perovskite solar cells (Sn-PSCs) without toxic lead ions outperform other types of lead-free PSCs in terms of photovoltaic performance. To avoid the oxidation of Sn2+ cations and the formation of vacancy defects, most reports involve the addition of SnF2 to the perovskite precursor solution, but hybrid tin halide (Sn-PVK) films still suffer from poor crystallinity and stability. In this work, we used an alternative additive of tin trifluoromethanesulfonate (Sn(OTF)2). Compared to SnF2, the solubility of Sn(OTF)2 in the precursor solution is greatly improved, and the crystal nucleation process is delayed, resulting in the enhancement of crystal growth. The coordination ability of the OTF- anions suppresses the oxidation of Sn2+ cations, which promotes the stability of Sn-PVK films. By replacing the conventional additive of SnF2 with Sn(OTF)2, the device achieves an increase in power conversion efficiency from 7.96% to 10.3%, while the stability of the devices is improved simultaneously.

Effect of mechanical stimulation on tissue heterotopic ossification: an in vivo experimental study.

Background: Heterotopic ossification of tendons and ligaments (HOTL) is a common clinical condition characterized by the absence of discernible features and a lack of effective treatment. In vitro experiments have demonstrated that mechanical stimulation can induce cell differentiation toward osteogenesis, thereby promoting heterotopic ossification. Currently, there are few experimental designs aimed at inducing ligament stretching in mice, and the mechanism of heterotopic ossification may not entirely mirror that observed in clinical cases. Therefore, there is an urgent imperative to develop a novel and feasible animal model. Methods: In this study, all the Enpp1 gene deficiency mice (a mouse model with heterotopic ossification of multiple ligaments) were divided into three groups: the control group, the spinal brake group, and the hyperactive group (treadmill training group). An external spinal fixation device was designed to restrict mice's spinal flexion and extension at 6 weeks of age. The brace was adjusted weekly according to the changes in the size of the mice. Additionally, treadmill training was used to increase activity in the spinal ligaments and Achilles tendons of the mice. Micro-CT scanning and HE staining were performed at 12, 20, and 28 W to evaluate the degree of ossification in the spinal ligament and Achilles tendon. What's more, As one of the mechanical stimulation transduction signals, YAP plays a crucial role in promoting osteogenic differentiation of cells. Immunofluorescence was utilized to assess YAP expression levels for the purpose of determining the extent of mechanical stimulation in tissues. Results: Our findings showed that a few ossification lesions were detected behind the vertebral space of mice at 8 weeks of age. Spinal immobilization effectively restricts the flexion and extension of cervical and thoracic vertebrae in mice, delaying spinal ligament ossification and reducing chronic secondary spinal cord injury. Running exercises not only enhance the ossification area of the posterior longitudinal ligament (PLL) and Achilles tendons but also exacerbate secondary spinal cord injury. Further immunofluorescence results revealed a notable increase in YAP expression levels in tissues with severe ossification, suggesting that these tissues may be subjected to higher mechanical stimulation. Conclusion: Mechanical stimulation plays a pivotal role in the process of heterotopic ossification in tissues. Our study provided valid animal models to further explore the pathological mechanism of mechanical stimulation in HOTL development.

Identification of novel gene signatures and immune cell infiltration in intervertebral disc degeneration using bioinformatics analysis.

Background: Intervertebral disc degeneration (IDD) is the leading cause of lower back pain, and an overall understanding of the molecular mechanisms related to IDD is still lacking. The purpose of this study was to explore gene signatures and immune cell infiltration related to IDD via bioinformatics analysis. Methods: A total of five expression profiles of mRNA and non-coding RNA were downloaded from the Gene Expression Omnibus (GEO) database. The potentially involved lncRNA/circRNA-miRNA-mRNA networks and protein-protein interaction networks were constructed by miRNet, circBank, STRING, and the Cytoscape database. Gene ontology, Kyoto Encyclopaedia of Genes and Genomes Analysis, Gene Set Enrichment Analysis, Gene Set Variation Analysis, Immune Infiltration Analysis, and Drug-Gene Interaction were used to analyse the top 20 hub genes. RT-qPCR was conducted to confirm the 12 differential expressions of genes both in the nucleus pulposus and annulus fibrosus tissues Results: There were 346 differentially expressed mRNAs, 12 differentially expressed miRNAs, 883 differentially expressed lncRNAs, and 916 differentially expressed circRNAs in the GEO database. Functional and enrichment analyses revealed hub genes associated with platelet activation, immune responses, focal adhesion, and PI3K-Akt signalling. The apoptotic pathway, the reactive oxygen species pathway, and oxidative phosphorylation play an essential role in IDD. Immune infiltration analysis demonstrated that the Treg cells had significant infiltration, and three levels of immune cells, including dendritic cells, Th2 cells, and tumour-infiltrating lymphocytes, were inhibited in IDD. Drug-gene interaction analysis showed that COL1A1 and COL1A2 were targeted by collagenase clostridium histolyticum, ocriplasmin, and PDGFRA was targeted by 66 drugs or molecular compounds. Finally, 24 cases of IDD tissues and 12 cases of normal disc tissues were collected, and the results of RT-qPCR were consistent with the bioinformatics results. Conclusion: Our data indicated that the 20 hub genes and immune cell infiltration were involved in the pathological process of IDD. In addition, the PDGFRA and two potential drugs were found to be significant in IDD development.

Efficacy and safety of novel biologics in the treatment of lupus nephritis based on registered clinical trials: a systematic review and network meta-analysis.

To compare the clinical effectiveness and safety of novel biologics for the treatment of lupus nephritis based on a reticulated meta-analysis approach. Registered clinical trials in 4 major databases (PubMed, Embase, Web of Science, The Cochrane Register of Clinical Trials) and ClinicalTrials.gov were systematically searched with a search time frame of build to June 2022. And we screened registered randomized controlled clinical trials of biologics for the treatment of lupus nephritis according to the protocol's nadir criteria. Trials were evaluated for quality using the Cochrane Risk of Bias Assessment Tool, and data were statistically analyzed using Stata 16.0 and Review Manager 5.3 software to compare and rank differences in effectiveness and safety between the biologics. A total of 10 registered randomized controlled clinical trials involving 2148 subjects were included in this study. The interventions were ranked from best to worst in terms of the primary outcome indicator of effectiveness, renal complete remission: belimumab > anifrolumab (900 + 300) mg > obinutuzumab > ocrelizumab 400 mg > abatacept 30/10 mg/kg > belimumab + rituximab > abatacept 10/10 mg/kg > abatacept (30/10 + 10/10) mg/kg > placeo > ocrelizumab 1000 mg > rituximab > anifrolumab 300 mg, belimumab was superior to placebo [OR = 1.75, 95% CI (1.13, 2.70)] and anifrolumab 300 mg [OR = 3.27, 95% CI (1.05, 10.14)], anifrolumab (900 + 300) mg was superior to anifrolumab 300 mg [OR = 3.56, 95% CI (1.30, 9.76)], and all were statistically significant. The ranking of each intervention in terms of overall renal remission for secondary outcome indicators from best to worst was: obinutuzumab > belimumab + rituximab > anifrolumab (900 + 300) mg > ocrelizumab 1000 mg > ocrelizumab 400 mg > belimumab > rituximab 1000 mg > abatacept 30/10 mg/kg > abatacept (30/10 + 10/10) mg/kg > placeo > abatacept 10/10 mg/kg > anifrolumab 300 mg, obinutuzumab was superior to placebo [OR = 2.27, 95% CI (1.11, 4.67)] and belimumab was also superior to placebo [OR = 1.56, 95% CI (1.07, 2.27)], and all were statistically significant. In terms of safety, with a focus on serious adverse events and serious infections, the results were: Serious adverse events at 1 year of monitoring occurred better with ocrelizumab 1000 mg than ocrelizumab 400 mg [OR = 0.51, 95% CI (0.29, 0.89)] and were statistically different; serious adverse events at 2 years of monitoring infection adverse events occurred better with obinutuzumab than with abatacept (30/10 + 10/10) mg/kg [OR = 0.24, 95% CI (0.07, 0.81)] and were statistically different. The safety of the new biologics in combination with conventional standard therapies is generally good, but it is belimumab and obinutuzumab that are most effective in achieving complete and overall remission in the kidney. This study protocol has been registered with PROSPERO, with a registration number of CRD42021262498.

DLX5 regulates the osteogenic differentiation of spinal ligaments cells derived from ossification of the posterior longitudinal ligament patients via NOTCH signaling.

Background: Ossification of the posterior longitudinal ligaments (OPLL) is common disorder characterized by heterotopic ossification of the spinal ligaments. Mechanical stimulation (MS) plays an important role in OPLL. DLX5 is an essential transcription factor required for osteoblast differentiation. However, the role of DLX5 during in OPLL is unclear. This study aims to investigate whether DLX5 is associated with OPLL progression under MS. Methods: Stretch stimulation was applied to spinal ligaments cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. Expression of DLX5 and osteogenesis-related genes were determined by quantitative real-time polymerase chain reaction and Western blot. The osteogenic differentiation ability of the cells was measured using alkaline phosphatase (ALP) staining and alizarin red staining. The protein expression of DLX5 in the tissues and the nuclear translocation of NOTCH intracellular domain (NICD) was examined by immunofluorescence. Results: Compared with non-OPLL cells, OPLL cells expressed higher levels of DLX5 in vitro and vivo (p < 0.01). Upregulated expression of DLX5 and osteogenesis-related genes (OSX, RUNX2, and OCN) were observed in OPLL cells induced with stretch stimulation and osteogenic medium, whereas there was no change in the non-OPLL cells (p < 0.01). Cytoplasmic NICD protein translocated from the cytoplasm to the nucleus inducing DLX5 under stretch stimulation, which was reduced by the NOTCH signaling inhibitors (DAPT) (p < 0.01). Conclusions: These data suggest that DLX5 play a critical role in MS-induced progression of OPLL through NOTCH signaling, which provides a new insight into the pathogenesis of OPLL.

Identification of immunodiagnostic blood biomarkers associated with spinal cord injury severity.

Blood always shows some immune changes after spinal cord injury (SCI), and detection of such changes in blood may be helpful for diagnosis and treatment of SCI. However, studies to date on blood immune changes after SCI in humans are not comprehensive. Therefore, to obtain the characteristics of blood immune changes and immunodiagnostic blood biomarkers of SCI and its different grades, a human blood transcriptome sequencing dataset was downloaded and analyzed to obtain differentially expressed immune-related genes (DEIGs), related functions and signaling pathways related to SCI and its various grades. Characteristic biomarkers of SCI and its different grades were identified by using weighted gene coexpression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) logistic regression. Expression of biomarkers was verified through experiments. The area under the curve (AUC) of biomarkers was calculated to evaluate their diagnostic value, and differences in immune cell content were examined. In this study, 17 kinds of immune cells with different contents between the SCI group and healthy control (HC) group were identified, with 7 immune cell types being significantly increased. Differences in the content of immune cells between different grades of SCI and the HC group were also discovered. DEIGs were identified, with alteration in some immune-related signaling pathways, vascular endothelial growth factor signaling pathways, and axon guidance signaling pathways. The SCI biomarkers identified and those of American Spinal Injury Society Impairment Scale (AIS) A and AIS D of SCI have certain diagnostic sensitivity. Analysis of the correlation of immune cells and biomarkers showed that biomarkers of SCI, AIS A grade and AIS D grade correlated positively or negatively with some immune cells. CKLF, EDNRB, FCER1G, SORT1, and TNFSF13B can be used as immune biomarkers for SCI. Additionally, GDF11and HSPA1L can be used as biomarkers of SCI AIS A grade; PRKCA and CMTM2 can be used as biomarkers of the SCI AIS D grade. Detecting expression of these putative biomarkers and changes in related immune cells may be helpful for predicting the severity of SCI.

Microwave-assisted extraction of bioactive components from Angelica dahurica using the tailor-made natural deep eutectic solvents.

In the present study, a new, green, efficient, and sustainable microwave-assisted extraction method combined with a deep eutectic solvent was successfully established for the extraction of five important coumarins from Angelica dahurica, namely bergapten, oxypeucedanin, imperatorin, cnidilin, and isoimperatorin. Compared with the conventional extraction method, the extraction efficiency of this method was improved by 10.74%. With increasingly serious global environmental pollution, this green method will be a solution for mainstream sustainable development and lead to a stable improvement in fine industries such as food, medicine, and cosmetics. The findings of this study may provide valuable clues and a scientific basis for further research of A. dahurica and other pharmaceutical components.

Increased blood flow of spinal cord lesion after decompression improves neurological recovery of degenerative cervical myelopathy: an intraoperative ultrasonography-based prospective cohort study.

INTRODUCTION: Surgical decompression is a highly effective therapy for degenerative cervical myelopathy (DCM), but the mechanisms of neurological recovery following decompression remain unclear. This study aimed to evaluate the spinal cord blood flow status after sufficient decompression by intraoperative contrast-enhanced ultrasonography (CEUS) and to analyze the correlation between neurological recovery and postdecompressive spinal cord blood perfusion in DCM. MATERIALS AND METHODS: Patients with multilevel DCM were treated by ultrasound-guided modified French-door laminoplasty using a self-developed rongeur. Neurological function was evaluated using the modified Japanese Orthopaedic Association (mJOA) score preoperatively and at 12 months postoperatively. Spinal cord compression and cervical canal enlargement before and after surgery were assessed by magnetic resonance imaging and computerized tomography. The decompression status was evaluated in real time by intraoperative ultrasonography, while the spinal cord blood flow after sufficient decompression was assessed by CEUS. Patients were categorized as favourable (≥50%) or unfavourable (<50%) recovery according to the recovery rate of the mJOA score at 12 months postoperatively. RESULTS: Twenty-nine patients were included in the study. The mJOA scores were significantly improved in all patients from 11.2±2.1 preoperatively to 15.0±1.1 at 12 months postoperatively, with an average recovery rate of 64.9±16.2%. Computerized tomography and intraoperative ultrasonography confirmed adequate enlargement of the cervical canal and sufficient decompression of the spinal cord, respectively. CEUS revealed that patients with favourable neurological recovery had a greater increased blood flow signal in the compressive spinal cord segment after decompression. CONCLUSIONS: In DCM, intraoperative CEUS can clearly reflect spinal cord blood flow. Patients with increased blood perfusion of the spinal cord lesion immediately after surgical decompression tended to achieve greater neurological recovery.

Inhibiting tau protein improves the recovery of spinal cord injury in rats by alleviating neuroinflammation and oxidative stress.

After spinal cord injury, the concentrations of total and hyperphosphorylated tau in cerebrospinal fluid increase, and levels of both correlate with injury severity. Tau inhibition is considered effective therapy for many central nervous system diseases, including traumatic brain injury and Alzheimer's disease. However, whether it can play a role in the treatment of spinal cord injury remains unclear. In this study, the therapeutic effects of tau inhibition were investigated in a rat model of transection spinal cord injury by injecting the rats with a lentivirus encoding tau siRNA that inhibits tau expression. We found that tau inhibition after spinal cord injury down-regulated the levels of inflammatory mediators, including tumor necrosis factor-α, interleukin-6 and interleukin-1ß. It also led to a shift of activated microglial polarization from the M1 pro-inflammatory phenotype to the M2 anti-inflammatory phenotype, and reduced the amount of reactive oxygen species in the acute phase. Furthermore, the survival of residual neural cells around the injury epicenter, and neuronal and axonal regeneration were also markedly enhanced, which promoted locomotor recovery in the model rats. Collectively, our findings support the conclusion that tau inhibition can attenuate neuroinflammation, alleviate oxidative stress, protect residual cells, facilitate neurogenesis, and improve the functional recovery after spinal cord injury, and thus suggest that tau could be a good molecular target for spinal cord injury therapy.

Deciphering postnatal limb development at single-cell resolution.

The early postnatal limb developmental progression bridges embryonic and mature stages and mirrors the pathological remodeling of articular cartilage. However, compared with multitudinous research on embryonic limb development, the early postnatal stage seems relatively unnoticed. Here, a systematic work to portray the postnatal limb developmental landscape was carried out by characterization of 19,952 single cells from murine hindlimbs at 4 postnatal stages using single-cell RNA sequencing technique. By delineation of cell heterogeneity, the candidate progenitor sub-clusters marked by Cd34 and Ly6e were discovered in articular cartilage and enthesis, and three cellular developmental branches marked by Col10a1, Spp1, and Tnni2 were reflected in growth plate. The representative transcriptomes and developmental patterns were intensively explored, and the key regulation mechanisms as well as evolvement in osteoarthritis were discussed. Above all, these results expand horizons of postnatal limb developmental biology and reach the interconnections between limb development, remodeling, and regeneration.

Inhibition of Neural Stem Cell Necroptosis Mediated by RIPK1/MLKL Promotes Functional Recovery After SCI.

Endogenous neural stem cells (eNSCs) are a new therapeutic strategy for the noninvasive repair of spinal cord injury (SCI). Necroptosis is a necrosome-dependent cell death process that serves as a significant regulatory mechanism in SCI. Current research shows that neurons, oligodendrocytes, and astrocytes all undergo necroptosis after SCI. However, it is unclear whether eNSCs are associated with necroptosis after SCI. By performing immunofluorescence analysis, we found that eNSCs undergo necroptosis during spinal cord injury repair in mice. Our present work demonstrates that receptor-interacting protein kinase 1 (RIPK1)/mixed lineage kinase domain-like protein (MLKL) are involved in necroptosis pathway in SCI mice. In vitro, the necroptosis induced by TNF-α/Smac-mimetic/Z-VAD-FMK (TSZ) treatment regulates phenotype of NSCs. In detail, the proliferative capacity of NSCs was significantly decreased in the presence of continual TSZ treatment, and the transcription of proinflammatory genes was upregulated, while the transcription of neurotrophic factors was inhibited. NSCs exhibited an obvious tendency to differentiate into glial cells under short-duration TSZ stimulation (6 h and 12 h); as the stimulus duration increased (24 h), the differentiation ability of the NSCs was significantly inhibited. These phenotypic changes are not conducive to neural cell survival and neural repair. Moreover, we examined the effect of necroptosis inhibitors on TSZ-treated NSCs. Necrostatin-1 and necrosulfonamide significantly reduced the necroptosis of NSCs after TSZ treatment and improved the phenotypic function of NSCs under TSZ stimulation. In additional in vivo experiments, after 2 weeks of administration, the necroptosis inhibitors reduced the necroptosis of NSCs and improved functional recovery in SCI mice. Taken together, these data indicate that the inhibition of NSC necroptosis with necroptosis inhibitors facilitates survival and phenotype maintenance in vitro and contributes to neuroprotection and repair in vivo. Our findings suggest that blocking necroptosis of eNSCs may be a potential therapeutic strategy for treating SCI.

A Novel Method of Making Hinges Using a Newly Designed Sharp Rongeur to Enhance Radiological and Clinical Outcomes in French-Door Cervical Expansive Laminoplasty.

OBJECTIVE: Although the lamina open angle of making hinges is closely related to the outcomes of French-door laminoplasty (FDL) for treatment of cervical spondylosis, there have been no methods to predict the lamina open angle preoperatively as yet. The aim of this study was to investigate the accuracy of predicting the laminal open angle using our newly designed sharp rongeur, and to compare the postoperative outcomes and complications between the methods of making hinges using the newly designed sharp rongeur and the traditional high-speed micro-drill during the FDL. METHODS: This was a single-center retrospective study. Following the approval of the institutional ethics committee, a total of 39 patients (Male: 28; Female: 11) diagnosed with cervical spondylos who underwent FDL in our institution between January 2018 and May 2019 were enrolled. Patients were divided into two groups based on the method of making hinges (sharp rongeur: 22 cases; high-speed micro-drill: 17 cases). The average age at surgery was 59.1 years (range: 16-85 years). The radiological parameters, clinical outcomes, modified Japanese Orthopaedic Association (mJOA) scale score, and the recovery rate of mJOA were recorded and compared between the groups, respectively. The radiological parameters and clinical measurements at pre- and post-operation stages were compared using the paired-sample t-test, the Wilcoxon signed-rank test, and the Friedman's test, and variables in the two groups were analyzed using an unpaired Student's t-test or a Mann-Whitney U test. RESULTS: The average follow-up period was 20.4 months (range: 14.0-25.9 months), the postoperative open angle was 60.13° ± 3.69° in the rongeur group with 22.78° ± 4.34° of angular enlargement, which was significantly lower than that of 68.96° ± 1.00° in the micro-drill group with 32.75° ± 4.22° of angular enlargement (U = 19.000, p < 0.001). The rongeur group showed a higher fusion rate (34.1% vs 14.7%, χ2  = 11.340, p = 0.001), and a lower fracture rate of the lamina (7.8% vs 25.5%, χ2  = 14.185, p < 0.001) at 1-month post-surgery, compared to the micro-drill group. There were no significant differences in the clinical outcomes and postoperative complications between the two groups (p > 0.05), except in the recovery rate of mJOA scores (0.836 ± 0.138 vs 0.724 ± 0.180, U = 115.000, p = 0.042) and neck disability index (NDI) at the final follow-up (7.55 ± 10.65 vs 14.71 ± 8.72, U = 94.000, p = 0.008). CONCLUSIONS: The special sharp rongeur with a tip angle of 20° could be a preferred method to make hinges during FDL, which can predict the laminal open angle accurately and enlarge it to about 23°, thus reducing the fracture rate and accelerating the bony fusion of hinges compared with the outcomes of the traditional micro-drill method.

Transplanting neurofibromatosis-1 gene knockout neural stem cells improve functional recovery in rats with spinal cord injury by enhancing the mTORC2 pathway.

The poor survival and low efficiency of neuronal differentiation limits the therapeutic effects of transplanted neural stem cells in the treatment of spinal cord injury. Neurofibromatosis-1 (NF-1) is a tumor suppressor gene that restricts the rapid and abnormal growth and differentiation of neural cells. In the present study, lentiviral vectors were used to knock out NF-1, Ricotr (the core member of mTORC2) or NF-1+Ricotr in neural stem cells in vitro, and the NF-1, Ricotr or NF-1+Ricotr knockout neural stem cells were transplanted at the lesion site in a rat model of spinal cord injury (SCI). We first demonstrated that targeted knockout of NF-1 had an antiapoptotic effect and improved neuronal differentiation by enhancing the mTORC2/Rictor pathway of neural stem cells in vitro. Subsequently, transplanting NF-1 knockout neural stem cells into the injured site sufficiently promoted the tissue repair and functional recovery of rats with spinal cord injury by enhancing the survival and neuronal differentiation of grafted neural stem cells. Collectively, these findings reveal a prominent role of NF-1 in neural stem cell biology, which is an invaluable step forward in enhancing the benefit of neural stem cell-mediated regenerative cell therapy for spinal cord injury and identifies the transplantation of NF-1 knockout neural stem cells as a promising strategy for spinal cord injury.

Integrative Bioinformatics Analysis Revealed Mitochondrial Dysfunction-Related Genes Underlying Intervertebral Disc Degeneration.

Objective: Mitochondrial dysfunction plays an important role in intervertebral disc degeneration (IDD). We aim to explore the pathways and key genes that cause mitochondrial dysfunction during IDD and to further reveal the pathogenesis of IDD based on bioinformatic analyses. Methods: Datasets GSE70362 and GSE124272 were downloaded from the Gene Expression Omnibus. Differentially expressed genes (DEGs) of mitochondrial dysfunction between IDD patients and healthy controls were screened by package limma package. Critical genes were identified by adopting gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) pathways, and protein-protein interaction (PPI) networks. We collected both degenerated and normal disc tissues obtained surgically, and we performed western blot and qPCR to verify the key DEGs identified in intervertebral disc tissues. Results: In total, 40 cases of IDD and 24 healthy controls were included. We identified 152 DEGs, including 67 upregulated genes and 85 downregulated genes. Four genes related to mitochondrial dysfunction (SOX9, FLVCR1, NR5A1 and UCHL1) were screened out. Of them, SOX9, FLVCR1, and UCHL1 were down-regulated in peripheral blood and intervertebral disc tissues of IDD patients, while NR5A1 was up-regulated. The analysis of immune infiltration showed the concentrations of mast cells activated were significantly the highest in IDD patients. Compared with the control group, the level of T cells CD4 memory resting was the lowest in the patients. In addition, 24 cases of IDD tissues and 12 cases of normal disc tissues were obtained to verify the results of bioinformatics analysis. Both western blot and qPCR results were consistent with the results of bioinformatics analysis. Conclusion: We identified four genes (SOX9, FLVCR1, NR5A1 and UCHL1) associated with mitochondrial dysfunction that play an important role in the progress of disc degeneration. The identification of these differential genes may provide new insights for the diagnosis and treatment of IDD.

Identification of hub genes related to the innate immune response activated during spinal cord injury.

Spinal cord injury (SCI) often leads to sensory and motor dysfunction. Two major factors that hinder spinal cord repair are local inflammation and glial scar formation after SCI, and thus appropriate immunotherapy may alleviate damage. To characterize changes in gene expression that occur during SCI and thereby identify putative targets for immunotherapy, here we analyzed the dataset GSE5296 (containing one control group and six SCI groups at different timepoints) to identify differentially-expressed genes. Functional enrichment analysis was performed and a protein-protein interaction network was created to identify possible hub genes. Finally, we performed quantitative PCR to verify changes in gene expression. The CIBERSORT algorithm was used to analyze innate immune cell infiltration patterns. The dataset GSE162610 (containing one control group and three SCI groups at different timepoints) was analyzed to evaluate innate immune cell infiltration at the single-cell level. The dataset GSE151371 (containing one control group [n = 10] and an SCI group [n = 38]) was used to detect the expression of hub genes in the blood from SCI patients. Differentially-expressed innate immune-related genes at each timepoint were identified, and the functions and related signaling pathways of these genes were examined. Six hub genes were identified and verified. We then analyzed the expression characteristics of these hub genes and characteristics of innate immune infiltration in SCI; finally, we examined ligand expression in the context of the CCL signaling pathway and COMPLEMENT signaling pathway networks. This study reveals the characteristics of innate immune cell infiltration and temporal expression patterns of hub genes, and may aid in the development of immunotherapies for SCI.

Comparative intra- and inter-observer reliability of two methods for evaluating intraoperative ultrasonography-based spinal cord hyperechogenicity intensity in degenerative cervical myelopathy.

OBJECTIVES: During French-door laminoplasty, a linear array transducer of IOUS was used to observe and record the spinal cord decompression. To acquire a higher-reliability method, and compare the in-observer and inter-observer reliability of two methods in evaluating the hyperechoic intensity of spinal cord ultrasound in degenerative cervical myelopathy (DCM). BACKGROUND: The intensity of spinal cord hyperechogenicity is considered as a potential predictor of neurological recovery in DCM after decompression, but the accuracy of gray value ratio (GVR) is affected by many factors. METHODS: Totally 28 patients (20 males and 8 females) who had been followed up for 12 months were included. Their mean age at surgery was 61.2 ± 10.8 years and the average symptom duration was 23.36 ± 22.11 months. The gray values of circles 1, 2 and 3 were recorded as Gcompression, Gnorml and Gsac, respectively. Circle 1 was drawn with the maximum brightness point within the spinal cord as the center, circle 2 with the same area was plotted on the spinal cord with uniform echogenicity, without compression and at least 1 cm away from the circle 1, and circle 3 was drawn on the dorsal dural sac at the same segment as circle 1. GVR was calculated as follows: GVR-A = Gcompression/Gnorml (method A), and GVR-B = Gcompression/Gsac (method B). The in-observer and inter-observer reliabilities of the two methods were compared. It is generally believed a reliability coefficient < 0.40 and > 0.75 indicate poor and good reliability respectively. The images-based GVR-B using this protocol demonstrates higher inter- and intraobserver reliabilities than GVR-A, and can be used as the basis for prognostic prediction and future studies. RESULTS: All examination acquisitions were successfully completed. GVR-A averaged 2.043 (0.318-5.56), and GVR-B averaged 0.578(0.06-1.41). GVR-B has better repeatability of gray value measurement, smaller relative standard deviation (RSD%) (0.298 vs. 0.32) and larger inter-group correlation coefficient compared with GVR-A. The mean value (MD) of the GVR difference calculated by GVR-B between the two clinicians was closer to 0. CONCLUSIONS: For DCM patients routinely using ultrasound for real-time cord visualization during spinal cord decompression by French-door laminoplasty, the images-based GVR-B using this protocol demonstrates better inter- and intraobserver reliabilities compared with GVR-A.

Dynamic elastic modulus assessment of the early degeneration model of an intervertebral disc in cynomolgus monkeys with one strike loading.

OBJECTIVE: Disc degeneration has long been associated with excessive mechanical loading or acute disc injury. Our goal is to perform a shock load on the functional units of the cynomolgus monkey intervertebral disc and analyze the degree of degeneration of the intervertebral disc through image analysis and comprehensive analysis. The organ model establishes a standard organ culture model and a non-invasive biomechanical evaluation protocol close to the early degeneration of the human intervertebral disc. METHODS: After modeling, the cynomolgus monkey intervertebral discs were collected and loaded into the dynamic mechanical culture system. The physiological group was loaded with 10% high compressive deformation load for one second, the injury group was punctured with annulus fibrosus, the model group was loaded with 20-50% high compressive deformation, and the nutritional components were a high-glucose group and low-glucose group. After day 3 (short term) and day 10 (long term), samples were collected to analyze cell viability, histomorphology, image analysis for imaging and biomechanical changes. RESULTS: Both the injury group and the 30-50% strain model group showed signs of early degeneration, including decreased instantaneous compressive stiffness, percent change in gray value, decreased cell viability, AF fissure, and percent increase in dynamic elastic modulus. The glucose-restricted group also showed signs of early disc degeneration in long-term cultures. CONCLUSION: This study shows that a single shock load can induce early degeneration of healthy cynomolgus monkey intervertebral discs, and 30% strain may be the nociceptive threshold for early degeneration of healthy intervertebral discs. More importantly, a non-invasive biomechanical evaluation scheme of Percentage change in dynamic modulus of elasticity is established, which solves the key scientific problem of how to non-invasively, quantitatively and sensitively detect the development process of early intervertebral disc degeneration and its degree of degeneration in an in vitro organ model.

Systematic study of single-cell isolation from musculoskeletal tissues for single-sell sequencing.

BACKGROUND: The single-cell platform provided revolutionary way to study cellular biology. Technologically, a sophistic protocol of isolating qualified single cells would be key to deliver to single-cell platform, which requires high cell viability, high cell yield and low content of cell aggregates or doublets. For musculoskeletal tissues, like bone, cartilage, nucleus pulposus and tendons, as well as their pathological state, which are tense and dense, it's full of challenge to efficiently and rapidly prepare qualified single-cell suspension. Conventionally, enzymatic dissociation methods were wildly used but lack of quality control. In the present study, we designed the rapid cycling enzymatic processing method using tissue-specific enzyme cocktail to treat different human pathological musculoskeletal tissues, including degenerated nucleus pulposus (NP), ossifying posterior longitudinal ligament (OPLL) and knee articular cartilage (AC) with osteoarthritis aiming to rapidly and efficiently harvest qualified single-cell suspensions for single-cell RNA-sequencing (scRNA-seq). RESULTS: We harvested highly qualified single-cell suspensions from NP and OPLL with sufficient cell numbers and high cell viability using the rapid cycling enzymatic processing method, which significantly increased the cell viability compared with the conventional long-time continuous digestion group (P < 0.05). Bioanalyzer trace showed expected cDNA size distribution of the scRNA-seq library and a clear separation of cellular barcodes from background partitions were verified by the barcode-rank plot after sequencing. T-SNE visualization revealed highly heterogeneous cell subsets in NP and OPLL. Unfortunately, we failed to obtain eligible samples from articular cartilage due to low cell viability and excessive cell aggregates and doublets. CONCLUSIONS: In conclusion, using the rapid cycling enzymatic processing method, we provided thorough protocols for preparing single-cell suspensions from human musculoskeletal tissues, which was timesaving, efficient and protective to cell viability. The strategy would greatly guarantee the cell heterogeneity, which is critical for scRNA-seq data analysis. The protocol to treat human OA articular cartilage should be further improved.