Single-cell analysis of spinal cord injury reveals functional heterogeneity of oligodendrocyte lineage cells.

Spinal cord injury (SCI) is a traumatic condition that causes myelin destruction and neuronal death, making it challenging to reverse. In spinal cord tissue, oligodendrocyte progenitor cells and oligodendrocytes are essential for maintaining myelin morphology and axon regeneration. The decrease in oligodendrocyte lineage cells after SCI is a major factor contributing to the difficulty in restoring spinal cord function. However, there is still a lack of research on the status and intercellular communication between oligodendrocyte lineage cells after injury. The development of single-cell sequencing technology has enabled researchers to obtain highly accurate cellular transcriptional information, facilitating detailed studies of cellular subpopulations. This study delved into the cellular heterogeneity of oligodendrocyte lineage cells using a single-cell transcriptomic approach to uncover functional changes and cellular interactions during different time points after SCI. Our findings highlighted the critical roles of Psap (Prosaposin)/Gpr37l1 and Psap/Gpr37 ligand-receptor pairs among oligodendrocyte lineage cells. Furthermore, we predicted the transcription factors that may play a key regulatory role. We demonstrated for the first time that Junb acts almost exclusively in mature oligodendrocytes, which provides a potential target for the study of oligodendrocyte transcriptional mechanisms.

Clinical and Radiographic Features of the Atlantoaxial Dislocation Associated With Kashin-Beck Disease.

OBJECTIVES: Keshin-Beck disease (KBD) is a particular type of osteoarthritis that affects many joints. However, the deformity of atlantoaxial joint has been rarely reported in KBD, and therefore its clinical and radiograph features have not been identified. METHODS: We reviewed data in 14 patients who were diagnosed with atlantoaxial dislocation (AAD) in KBD at our institution. The demographic data, clinical history, imaging data, operative data, and Japanese Orthopaedic Association score were collected for evaluation. RESULTS: The mean age at presentation was 50 ± 1.7 years old. The most common features of AAD in KBD were the osteoarthritis, characterized by hypertrophic dens and anterior arch of the atlas. The average inner anteroposterior diameter (IAPD) of C1 was 28 ± 3.5 mm and the average spinal canal diameter was 14 ± 3.3 mm, which were respectively lower than the control level. Five patients had severe C1 stenosis (IAPD < 26mm). Separated odontoid process, like os odontoideum, was seen 9 patients. The tip of dens fused to C1 was observed in 4 patients; 12 patients had high-riding vertebral artery; and 5 patients had severe C1 stenosis, and they underwent C1 laminectomy with C1-C2 interarticular fusion or occipital-cervical fusion. All the patients displayed neurologic improvement after surgery. CONCLUSIONS: The atlantoaxial level could be affected by KBD, which may lead to typical abnormalities and cause AAD. A C1 laminectomy with an C1-C2 interarticular fusion or occipital-cervical fusion is recommended for the patient with severe stenosis.

Bioinformatic Data Mining for Candidate Drugs Affecting Risk of Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ) in Cancer Patients.

Background: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) leads to significant morbidity. Other coadministered drugs may modulate the risk for BRONJ. The present study aimed to leverage bioinformatic data mining to identify drugs that potentially modulate the risk of BRONJ in cancer. Methods: A GEO gene expression dataset of peripheral blood mononuclear cells related to BRONJ in multiple myeloma patients was downloaded, and differentially expressed genes (DEGs) in patients with BRONJ versus those without BRONJ were identified. A protein-protein interaction network of the DEGs was constructed using experimentally validated interactions in the STRING database. Overrepresented Gene Ontology (GO) molecular function terms and KEGG pathways in the network were analysed. Network topology was determined, and 'hub genes' with degree ≥2 in the network were identified. Known drug targets of the hub genes were mined from the 'drug gene interaction database' (DGIdb) and labelled as candidate drugs affecting the risk of BRONJ. Results: 751 annotated DEGs (log FC ≥ 1.5, p < 0.05) were obtained from the microarray gene expression dataset GSE7116. A PPI network with 633 nodes and 168 edges was constructed. Data mining for drugs interacting with 49 gene nodes was performed. 37 drug interactions were found for 9 of the hub genes including TBP, TAF1, PPP2CA, PRPF31, CASP8, UQCRB, ACTR2, CFLAR, and FAS. Interactions were found for several established and novel anticancer chemotherapeutic, kinase inhibitor, caspase inhibitor, antiangiogenic, and immunomodulatory agents. Aspirin, metformin, atrovastatin, thrombin, androgen and antiandrogen drugs, progesterone, Vitamin D, and Ginsengoside 20(S)-Protopanaxadiol were also documented. Conclusions: A bioinformatic data mining strategy identified several anticancer, immunomodulator, and other candidate drugs that may affect the risk of BRONJ in cancer patients.

Downregulated fat mass and obesity-associated protein inhibits bone resorption and osteoclastogenesis by nuclear factor-kappa B inactivation.

During osteoporosis, fat mass and obesity-associated protein (FTO) promotes the shift of bone marrow mesenchymal stem cells to adipocytes and represses osteoblast activity. However, the role and mechanisms of FTO on osteoclast formation and bone resorption remain unknown. In this study, we investigated the effect of FTO on RAW264.7 cells and bone marrow monocytes (BMMs)-derived osteoclasts in vitro and observed the influence of FTO on ovariectomized (OVX) mice model to mimic postmenopausal osteoporosis in vivo. Results found that FTO was up-regulated in BMMs from OVX mice. Double immunofluorescence assay showed co-localization of FTO with tartrate-resistant acid phosphatase (TRAP) in femurs of OVX mice. FTO overexpression enhanced TRAP-positive osteoclasts and F-actin ring formation in RAW264.7 cells upon RANKL stimulation. The expression of osteoclast differentiation-related genes, including nuclear factor of activated T cells c1 (NFATc1) and c-FOS, was upregulated in BMMs and RAW264.7 cells after FTO overexpression. FTO overexpression induced the phosphorylation and nuclear translocation of factor-kappa B (NF-κB) p65 in BMMs and RAW264.7 cells exposed to RANKL. ChIP and dual-luciferase assays revealed that FTO overexpression contributed to RANKL-induced binding of NF-κB to NFATc1 promoter. Rescue experiments suggested that FTO overexpression-mediated osteoclast differentiation was suppressed after intervention with a NF-κB inhibitor pyrrolidine dithiocarbamate. Further in vivo evidence revealed that FTO knockdown increased bone trabecula and bone mineral density, inhibited bone resorption and osteoclastogenesis in osteoporotic mice. Collectively, our research demonstrates that downregulated FTO inhibits bone resorption and osteoclastogenesis through NF-κB inactivation, which provides a novel reference for osteoporosis treatment.

Stromal cell-derived factor (SDF)-1α and platelet-rich plasma enhance bone regeneration and angiogenesis simultaneously in situ in rabbit calvaria.

The current study aimed to evaluate the effects of chemokine stromal cell-derived factor (SDF)-1α and platelet-rich plasma (PRP) on bone formation and angiogenesis, and to assess whether SDF-1α and PRP could function synergistically. Four evenly distributed defects (8 mm in diameter) were generated in the calvarial bones of New Zealand white rabbits. All rabbits received four treatment regimens containing autogenous bone particles (AB), SDF-1α, or PRP. AB group presented significantly less bone formation compared with the other three groups 2 and 4 weeks after surgery. The amount of newly formed bone in the AB+PRP+SDF-1α group was similar to that in the AB + SDF-1α group at the 4-week time-point (p = 0.65), and was much greater than that in the AB and AB+PRP group (p < 0.001). Meanwhile, more new blood vessels were formed in the AB+PRP, AB+SDF-1α, and AB+PRP+SDF-1α group versus the AB group. AB+PRP+SDF-1α group showed statistically increased angiogenesis compared with the AB+PRP and AB+SDF-1α groups (both p < 0.05) after treatment for 2 and 4 weeks. These findings indicated that SDF-1α and PRP might exhibit synergistic effects to promote angiogenesis in early bone regeneration.

Anti-human serum albumin autoantibody may be involved in the pathogenesis of autoimmune bullous skin diseases.

Although effective immunological diagnostic systems for autoimmune bullous skin diseases (AIBD) have been established, there are still unidentified cutaneous autoantigens. The purpose of this study is to investigative whether anti-human serum albumin (HSA) autoantibodies exist in AIBD sera and their potential pathogenesis. By immunoprecipitation-immunoblotting, immunofluorescence assay, anti-HSA autoantibodies could be detected in AIBD sera; by ELISAs, positive rates of AIBD sera for IgG and IgA anti-HSA autoantibodies were 29% and 34%, respectively. The IgG anti-HSA autoantibodies in ABID sera recognized a number of HSA antigen epitopes and therefore a polyclonal antibody against HSA were next employed to study its pathogenesis. In vitro cell and tissue culture models, anti-HSA antibody could influence DNA damage-related signaling proteins, via activation of phospho-p38 signaling pathway. This is the first report that an autoantibody may influence DNA damage-related signaling proteins. Statistical analyses also proved that anti-HSA autoantibodies were positively correlated with various known autoantibodies and clinical features of ABID patients. In summary, IgG and IgA autoantibodies to HSA may have diagnosis values for AIBD. DNA damage-related signaling proteins might be involved in the pathogenic role of anti-HSA autoantibodies in AIBD. Phospho-p38 signaling pathway is a potential target for treatment of AIBD positive for serum anti-HSA autoantibodies.

Synovial fibroblast-targeting liposomes encapsulating an NF-κB-blocking peptide ameliorates zymosan-induced synovial inflammation.

Synovial fibroblasts (SFs) play a crucial role in the inflammatory process of rheumatoid arthritis (RA). The highly activated NF-κB signal in SFs is responsible for most of the synovial inflammation associated with this disease. In this study, we have developed an SF-targeting liposomal system that encapsulates the NF-κB-blocking peptide (NBD peptide) HAP-lipo/NBD. HAP-lipo/NBDs demonstrated efficient SF-specific targeting in vitro and in vivo. Our study also showed a significant inhibitory effect of HAP-lipo/NBD on NF-κB activation, inflammatory cytokine release and SF migration capability after zymosan stimulation. Furthermore, the systemic administration of HAP-lipo/NBDs significantly inhibited synovial inflammation and improved the pathological scores of arthritis induced by zymosan. Thus, these results suggest that an SF-targeting NF-κB-blocking strategy is a potential approach for the development of alternative, targeted anti-RA therapies.

Modification of Human Umbilical Cord Blood Stem Cells Using Polyethylenimine Combined with Modified TAT Peptide to Enhance BMP-2 Production.

With the emerging role of umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) for bone regeneration and delivery of therapeutic proteins, there is an increasing need for effective gene delivery systems to modify such cells. mTAT, a TAT peptide sequence bearing histidine and cysteine residues, has been successfully used for intracellular gene delivery. Using a gWiz-GFP plasmid, we demonstrated that polyethylenimine combined with mTAT (mTAT/PEI) displayed good transfection efficacy in hUCB-MSC. hUCB-MSC transfected with mTAT/PEI were shown to express more BMP-2 protein and mRNA, indicating the feasibility of using the cells as a BMP-2 delivery system. Importantly, compared to PEI25, a "gold standard" nonviral transfection polymer, mTAT/PEI had limited toxicity to the cells. Furthermore, we demonstrated enhanced osteogenic activity in vitro for BMP-2 expressing hUCB-MSC. These results provide encouraging evidence for the potential use of mTAT/PEI to genetically modify hUCB-MSC as an approach to enhance tissue regeneration.

Effects of unfractionated heparin and rivaroxaban on the expression of heparanase and fibroblast growth factor 2 in human osteoblasts.

Long-term treatment with anticoagulants may contribute to osteoporosis. Although unfractionated heparin and rivaroxaban have adverse effects on bone microstructure and function in adult rats, the underlying mechanism remains to be elucidated. Heparanase (HPSE) and fibroblast growth factor (FGF)2 are important signals in bone formation and fracture healing. Therefore, the present study was designed to investigate the effects of unfractionated heparin and rivaroxaban on the expression of HPSE and FGF2 in human osteoblasts. Human osteoblasts were treated with unfractionated heparin (0.5-50 IU/ml) or rivaroxaban (0.13­13 µg/ml) for different durations. Plasmids encoding HPSE and FGF2 were transfected into osteoblasts, and cell viability was assessed using MTT assays, with mRNA and protein expression levels determined using reverse transcription­quantitative polymerase chain reaction and western blot analyses, respectively. Osteoblast growth was significantly inhibited by treatment with unfractionated heparin (50 IU/ml) or rivaroxaban (13 µg/ml). Unfractionated heparin alone significantly inhibited the expression of HPSE and FGF2, whereas rivaroxaban inhibited the expression of FGF2 without affecting that of HPSE. Furthermore, the overexpression of HPSE or FGF2 significantly reversed the inhibitory effects of unfractionated heparin and rivaroxaban on osteoblasts. These findings suggested that HPSE and FGF2 signals were involved in the detrimental role of unfractionated heparin and rivaroxaban in human osteoblasts, providing novel information on the side effects of anticoagulants.

Comparison of the effects of heparin and the direct factor Xa inhibitor, rivaroxaban, on bone microstructure and metabolism in adult rats.

AIM: Deep venous thrombosis is a significant complication following surgery, and is associated with high morbidity and mortality in adults. The direct factor Xa inhibitor, rivaroxaban, is used to prevent venous thromboembolism in patients suffering from trauma and joint arthroplasty. The present study compared the effects of rivaroxaban and heparin on bone microstructure and metabolism in adult rats. MATERIALS AND METHODS: Twenty-four Wistar rats were divided into sham, rivaroxaban and heparin groups. Rivaroxaban (1.5 mg·kg(-1)·d(-1)) and heparin (2 IU·g(-1)·d(-1)) were administered for 4 weeks. To assess changes in bone metabolism, serum calcium and phosphorus levels, and bone formation and resorption markers were examined. Micro-CT analysis was used to examine the microstructure of both trabecular and cortical bone. Dual energy X-ray absorptiometry was employed to detect bone mineral density (BMD). RESULTS: Serum phosphorus levels were significantly lower in both rivaroxaban (1.33 ± 0.07 mmol/L) and heparin (1.33 ± 0.21 mmol/L) rats than in sham rats (1.71 ± 0.14 mmol/L). Activity and levels of bone formation markers, bone-specific alkaline phosphatase (BAP) and type I procollagen N-terminal pro-peptide (PINP), were 32.4 and 38.2% lower in heparin-treated rats than in sham rats. Bone resorption markers, pyridinoline (PYD) and deoxypyridinoline (DPD), were 20.1 and 34.3% higher in heparin-treated rats than in sham rats, respectively. By contrast, rivaroxaban only resulted in a decrease PINP levels. Bone volume fraction (BV/TV) decreased by 23.5 and 20.5% from those in sham rats, while trabecular separation (Tb.Sp) increased by 28.2 and 16.3% in trabecular bone of heparin- and rivaroxaban-treated rats, [corrected] respectively. Moreover, the microstructure of cortical bone and BMD were negatively affected by heparin but not by rivaroxaban. CONCLUSION: Rivaroxaban leads to fewer adverse effects on bone microstructure than heparin.

Matrine inhibits the growth and induces apoptosis of osteosarcoma cells in vitro by inactivating the Akt pathway.

Matrine, a natural product, has been demonstrated to be a promising chemotherapeutic drug for some cancers. Using flow cytometric analysis of the cell cycle and apoptosis, we found that matrine inhibited the proliferation and induced apoptosis in the human osteosarcoma (OS) cell lines MG63, HOS, U2OS, and SAOS2 in vitro in a dose-dependent manner. We therefore assessed the role of the serine/threonine kinase Akt in the regulation of matrine-mediated cell growth inhibition and apoptosis induction in human OS cell lines. After treatment for 48 h, matrine induced G0/G1-stage cell cycle arrest in MG63, U2OS, and SAOS2 cells associated with an increase in the expression of p27(Kip1) and a decrease in the expression of Akt, glycogen synthase kinase 3 (GSK3)-ß (Ser9), and cyclin D1. Furthermore, the pro-apoptotic factor Bax was upregulated. Overall, our findings suggest that matrine may be an effective anti-osteosarcoma drug due to its ability to inhibit proliferation and induce apoptosis in OS cells, possibly through the involvement of Akt signaling.

Curcumin improves the recovery of motor function and reduces spinal cord edema in a rat acute spinal cord injury model by inhibiting the JAK/STAT signaling pathway.

Curcumin, a yellow pigment extracted from Carcuma longa, has been demonstrated to have extensive pharmacological activity in various studies, and it exhibits protective effects on injuries involving a number of human organs. The present study was designed to evaluate the potential effect and underlying mechanism of curcumin on the motor function and spinal cord edema in a rat acute spinal cord injury (SCI) model. The SCI model was induced by a heavy object falling. At 30min after the SCI was successfully induced, the animals were intraperitoneally given 40mg/kg curcumin. The Basso, Beattie and Bresnahan scores showed that curcumin moderately improved the recovery of the motor function in the injured rats, and hematoxylin-eosin staining demonstrated the role of this compound in reducing the hemorrhage, edema and neutrophil infiltration of the traumatic spinal cord. Furthermore, curcumin also inhibited the SCI-associated aquaporin - 4 (AQP4) overexpression and glial fibrillary acidic protein (GFAP) and repressed the unusual activation of the JAK/STAT signaling pathway. In conclusion, our data demonstrate that curcumin exhibits a moderately protective effect on spinal cord injury, and this effect might be related to the inhibition of overexpressed AQP4 and GFAP and the activated JAK/STAT signaling pathway. Curcumin may have potential for use as a therapeutic option for spinal cord injuries.

Inhibition of focal adhesion kinase induces apoptosis in human osteosarcoma SAOS-2 cells.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase protein, acts as an early modulator of integrin signaling cascade, regulating basic cellular functions. In transformed cells, unopposed FAK signaling has been considered to promote tumor growth, progression, and metastasis. The aim of this study was to assess the role of focal adhesion kinase in human osteosarcoma SAOS-2 cells. SAOS-2 cells were transfected with PGPU6/GFP/shNC, and PGPU6/GFP/FAK-334 (shRNA-334), respectively. Expression of FAK was detected by real-time PCR and western blots. MTT assay was used to examine changes in cell proliferation. Cell apoptosis was analyzed by flow cytometry. The expression of caspase-3,-7,-9 was measured by Western blots. The expression of FAK in SAOS-2 cells significantly decreased in shRNA-334 group contrast to the control group (P < 0.01). Cells proliferation was inhibited by shRNA-334 and shRNA-334 + cisplatin, and the effects were clearly enhanced when cells treated with the anticancer agents. The level of cell apoptosis in shRNA-334 and shRNA-334 + cisplatin group was higher than in the control group (P < 0.01). The current data support evidence that down-regulation of FAK could induce SAOS-2 apoptosis through the caspase-dependent cell death pathway. Inhibition of the kinases may be important for therapies designed to enhance the apoptosis in osteosarcoma.

Curcumin promotes the spinal cord repair via inhibition of glial scar formation and inflammation.

Spinal cord injury (SCI) is a serious clinical situation without any effective therapy to date. Traumatic SCI triggers a complex pathological process including inflammatory response and glial scar formation. In this study, we demonstrated that curcumin, a natural product which functions as an anti-inflammatory agent, inhibited the activation of signal transducer and activator of transcription-3 and NF-kappa B in the injured spinal cord. Curcumin treatment greatly reduced the astrogliosis in SCI mice and significantly decreased the expression of IL-1ß and NO, as well as the number of Iba1(+) inflammatory cells at the lesion site. Notably, more residual axons and neurons were protected and significantly improved functional recovery was observed in the curcumin-treated mice, compared to the mice without curcumin treatment. These findings indicate that curcumin promotes spinal cord repair through inhibiting glial scar formation and inflammation and suggests the therapeutic potential of curcumin for SCI.

[Clinical application of a novel posterior lumbar fusion method and the short-term observation of its effect].

OBJECTIVE: To increase local blood supply of bone graft, a novel posterior lumbar spine fusion method with orthotopic paraspinal muscle-pediculated bone flaps was constructed, and the fusion rate and clinical effect.were observed. METHODS: From June 2007 to December 2010, 117 patients of lumbar spinal stenosis or lumbar destabilization treated with the novel posterior lumbar fusion method were studied, 49 males and 68 females, aged from 40 to 77 years, average 61.5 years. Clinical effect was evaluated by JOA and VAS score preoperatively and postoperatively, and the fusion result was evaluated by three-dimensional CT reconstruction postoperatively. RESULTS: Seventeen cases lost of follow up, the rest were followed up from 7 to 38 months, average 19 months. There was significant difference between pre- and postoperative JOA and VAS score (P < 0.01), the preoperative JOA score was 10.3 ± 1.9, and 25.4 ± 4.2 at the latest follow-up, the improvement rate was 81.0% ; the preoperative VAS score was 8.5 ± 0.8, and 2.3 ± 0.4 at the latest follow-up. The three-dimensional CT reconstruction showed that 126 of the 133 segments formed solid fusion in 100 patients who completed the follow-up, the fusion rate was 94.7%. CONCLUSION: The novel posterior lumbar fusion method make the bone graft position more precise, stable and increases the fusion rate, which can effectively reduce pseudarthrosis and have a promising clinical effect.

Viability inhibition effect of gambogic acid combined with cisplatin on osteosarcoma cells via mitochondria-independent apoptotic pathway.

We previously demonstrated that gambogic acid (GA) is a promising chemotherapeutic compound for human osteosarcoma treatment. The aim of this study was to detect whether the combination of lower-dose GA (0.3 mg/L) and cisplatin (CDDP) (1 mg/L) could perform a synergistic effect on inhibiting tumor in four osteosarcoma cell lines. Our results showed that the combination between GA at lower dose and CDDP significantly exerts a synergistic effect on inhibiting the cellular viability in MG63, HOS, and U2OS cells. In contrast, an antagonistic character was detected in SAOS2 cells exposed to the combined use of lower-dose GA (0.3 mg/L) and CDDP (1 mg/L). Then, analysis of cell cycle showed the combination of both drugs significantly induced the G2/M phase arrest, without any difference relative to GA treatment alone, in MG63 cells. Flow-cytometric analysis of cell apoptosis displayed that the apoptotic rate in the combination group is higher than that in GA treatment alone in MG63, HOS, and U2OS cells. The combined use of both drugs had no effect on mitochondrial membrane potential, but promoted the apoptosis-inducing function through triggering of CDDP in the three cell lines. By measurement of mitochondrial membrane potential, the activity of caspase-3 and the expressions of caspase-8 and caspase-9, it was showed that the apoptosis-promoting effect of the combined use of both drugs could be dependent on the death receptor apoptosis pathway, not dependent on the mitochondria apoptosis mechanism. This research, for the first time, demonstrates that GA could increase the chemotherapeutic effect of CDDP in human osteosarcoma treatment through inducing the cell cycle arrest and promoting cell apoptosis.