Mesenchymal stem cell exosomes inhibit nucleus pulposus cell apoptosis via the miR-125b-5p/TRAF6/NF-κB pathway axis.

Intervertebral disc degeneration (IVDD) is the pathological basis of a range of degenerative spinal diseases and is the primary cause of lower back pain. Mesenchymal stem cell (MSC) transplantation inhibits IVDD progression. However, the specific mechanisms that underlie these effects remain unclear. In this study, candidate microRNAs (miRNAs) are screened using bioinformatics and high-throughput sequencing. TNF-α is used to induce nucleus pulposus cell (NPC) degeneration. MSC-derived exosomes (MSC-exosomes) are obtained using high-speed centrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis. Cell viability is determined by CCK-8 assay. Flow cytometry and TUNEL assays are used to detect cell apoptosis. The expression levels of miR-125b-5p are detected by RT-qPCR, and a dual-luciferase gene reporter assay confirms the downstream target genes of miR-125b-5p. Protein expression is determined by western blot analysis. Rat models are used to validate the function of miR-125b-5p in MSC-exosomes. The results show that miR-125b-5p is expressed at low levels in degenerated disc tissues compared with that in normal disc tissues; however, it is highly expressed in MSC-exosomes. Furthermore, MSC-exosomes are efficiently taken up by NPCs while miR-125b-5p is delivered into NPCs; thus, MSC-exosomes act as inhibitors of apoptosis in NPCs. Overexpression of miR-125b-5p downregulates TRAF6 expression and inhibits NF-κB activation. However, TRAF6 overexpression reverses these effects of miR-125b-5p. We demonstrate that MSC-exosomes attenuate IVDD in vivo by delivering miR-125b-5p. MSC-exosomes can deliver miR-125b-5p to target TRAF6, inhibit NF-κB activation, and attenuate the progression of IVDD.

TCF7/SNAI2/miR-4306 feedback loop promotes hypertrophy of ligamentum flavum.

BACKGROUND: Hypertrophy of ligamentum flavum (HLF) is the mainly cause of lumbar spinal stenosis (LSS), but the precise mechanism of HLF formation has not been fully elucidated. Emerging evidence indicates that transcription factor 7 (TCF7) is the key downstream functional molecule of Wnt/ß-catenin signaling, which participated in regulating multiple biological processes. However, the role and underlying mechanism of TCF7 in HLF is still unclear. METHODS: We used mRNAs sequencing analysis of human LF and subsequent confirmation with RT-qPCR, western blot and immunohistochemistry to identified the TCF7 in HLF tissues and cells. Then effect of TCF7 on HLF progression was investigated both in vitro and in vivo. Mechanically, chromatin immunoprecipitation, dual-luciferase reporter assays, and rescue experiments were used to validate the regulation of TCF7/SNAI2/miR-4306 feedback loop. RESULTS: Our results identified for first time that the TCF7 expression was obviously elevated in HLF tissues and cells compared with control, and also found that TCF7 expression had significant positive correlation with LF thickness and fibrosis score. Notably, TCF7 inhibition suppressed the hyper-proliferation and fibrosis phenotype of HLF cells in vitro and ameliorated progression of HLF in mice in vivo, whereas TCF7 overexpression promoted hyper-proliferation and fibrosis phenotype of HLF cells in vitro. Our data further revealed that TCF7 interacted with SNAI2 promoter to transactivated the SNAI2 expression, thereby promoting hyper-proliferation and fibrosis phenotype of HLF cells in vitro. Furthermore, miR-4036 negatively regulated by SNAI2 could negatively feedback regulate TCF7 expression by directly binding to TCF7 mRNA 3'-UTR, thus inhibiting the hyper-proliferation and fibrosis phenotype of HLF cells in vitro. CONCLUSIONS: Our study demonstrated that TCF7 inhibition could suppress HLF formation by modulating TCF7/SNAI2/miR-4306 feedback loop, which might be considered as a novel potential therapeutic target for HLF.

Absence of VEGFR-1/Flt-1 signaling pathway in mice results in insensitivity to discogenic low back pain in an established disc injury mouse model.

Although degenerative disc disease (DDD) and related low back pain (LBP) are growing public health problems, the underlying disease mechanisms remain unclear. An increase in the vascular endothelial growth factor (VEGF) levels in DDD has been reported. This study aimed to examine the role of VEGF receptors (VEGFRs) in DDD, using a mouse model of DDD. Progressive DDD was induced by anterior stabbing of lumbar intervertebral discs in wild type (WT) and VEGFR-1 tyrosine-kinase deficient mice (vegfr-1TK-/- ). Pain assessments were performed weekly for 12 weeks. Histological and immunohistochemical assessments were made for discs, dorsal root ganglions, and spinal cord. Both vegfr-1TK-/- and WT mice presented with similar pathological changes in discs with an increased expression of inflammatory cytokines and matrix-degrading enzymes. Despite the similar pathological patterns, vegfr-1TK-/- mice showed insensitivity to pain compared with WT mice. This insensitivity to discogenic pain was related to lower levels of pain factors in the discs and peripheral sensory neurons and lower spinal glial activation in the vegfr-1TK- /- mice than in the WT mice. Exogenous stimulation of bovine disc cells with VEGF increased inflammatory and cartilage degrading enzyme. Silencing vegfr-1 by small-interfering-RNA decreased VEGF-induced expression of pain markers, while silencing vegfr-2 decreased VEGF-induced expression of inflammatory and metabolic markers without changing pain markers. This suggests the involvement of VEGFR-1 signaling specifically in pain transmission. Collectively, our results indicate that the VEGF signaling is involved in DDD. Particularly, VEGFR-1 is critical for discogenic LBP transmission independent of the degree of disc pathology.

Hypoxia reduces the osteogenic differentiation of peripheral blood mesenchymal stem cells by upregulating Notch-1 expression.

Purpose: Mesenchymal stem cells (MSCs) seeded on biocompatible scaffolds have therapeutic potential for bone defect repair. However, MSCs can be affected by hypoxia and nutritional deficiency due to a lack of blood vessels in the scaffolds. Here, we explored the effects of hypoxia on MSC differentiation to clarify these mechanisms. Methods: Peripheral blood mesenchymal stem cells (PBMSCs) were cultured in small individual chambers with oxygen concentrations of 1%, 9%, and 21%. Cell proliferation was evaluated by Cell Counting Kit 8 assays, and cell survival was determined using live/dead assays. Scratch assays were performed to evaluate cell migration. Ca2+ deposition/mineralization experiments, reverse transcription quantitative real-time polymerase chain reaction, and Western blotting were performed to assess the osteogenic differentiation of cells. Notch1 expression was downregulated by lentivirus-transfected PBMSCs to observe the effects of Notch1 knockdown on osteogenic gene and protein expression. Results: PBMSCs exposed to hypoxia (1% O2) demonstrated accelerated proliferation, increased migration, and reduced survival in the absence of serum. Although 9% oxygen promoted osteogenic differentiation, the osteogenic differentiation of PBMSCs was significantly reduced by 1% O2, and this effect was associated with increased Notch1 expression. Reducing Notch1 expression using small interfering RNA significantly restored the osteogenic differentiation of PBMSCs. Conclusions: Hypoxia accelerated proliferation, increased migration, and reduced PBMSC differentiation into osteoblasts by increasing Notch1 expression. These findings may contribute to the development of appropriate cell culture or in vivo transplantation conditions to maintain the full osteogenic potential of PBMSCs.

Anatomic morphology and clinical significance of intraforaminal ligaments of the cervical spine.

The cause of cervical spondylotic radiculopathy could be related to the intraforaminal ligaments (IFLs) of the cervical spine. The aim of this study is to identify and describe the IFLs and assess their clinical significance. Six intact cervical spine specimens from adult embalmed cadavers were dissected to expose the cervical nerve roots and their surrounding intraforaminal tissues fully. From the C1-C2 to the C7-T1 intervertebral foramina (IVF), the connective structures between each nerve root and its surrounding foraminal wall were examined under a surgical microscope. The morphology, number, and attachment points of the IFLs of each segment were documented, and the length, width, or diameter and thickness of the ligaments were measured with a vernier caliper. IFLs were observed in all 84 IVFs of the cervical spine. According to their locations, they can be divided into two categories: the first is entrance-zone IFLs, which are radially distributed around the nerve root; the second is mid-zone IFLs, which are thin, strip-shaped fibrous tissues intertwined around the nerve roots, the number of ligaments being considerable but difficult to quantify. Ligament structures have been identified in the IVF of the cervical spine. Under physiological conditions, they could be protective in maintaining the position, shape, and function of nerve roots. However, under pathological conditions, the IFLs of the cervical spine could aggravate the symptoms of cervical nerve root radicular pain associated with other types of compression. Clin. Anat. 32:654-660, 2019. © 2019 Wiley Periodicals, Inc.

Development of an in vivo mouse model of discogenic low back pain.

Discogenic low back pain (DLBP) is extremely common and costly. Effective treatments are lacking due to DLBP's unknown pathogenesis. Currently, there are no in vivo mouse models of DLBP, which restricts research in this field. The aim of this study was to establish a reliable DLBP model in mouse that captures the pathological changes in the disc and allows longitudinal pain testing. The model was generated by puncturing the mouse lumbar discs (L4/5, L5/6, and L6/S1) and removing the nucleus pulposus using a microscalpel under the microscope. Histology, molecular pathways, and pain-related behaviors were examined. Over 12 weeks post-surgery, animals displayed the mechanical, heat, and cold hyperalgesia along with decreased burrowing and rearing. Histology showed progressive disc degeneration with loss of disc height, nucleus pulposus reduction, proteoglycan depletion, and annular fibrotic disorganization. Immunohistochemistry revealed a substantial increase in inflammatory mediators at 2 and 4 weeks. Nerve growth factor was upregulated from 2 weeks to the end of the experiment. Nerve fiber ingrowth was induced in the injured discs after 4 weeks. Disc-puncture also produced an upregulation of neuropeptides in dorsal root ganglia neurons and an activation of glial cells in the spinal cord dorsal horn. These findings indicate that the cellular and structural changes in discs, as well as peripheral and central nervous system plasticity, paralleled persistent, and robust behavioral pain responses. Therefore, this mouse DLBP model could be used to investigate mechanisms underlying discogenic pain, thereby facilitating effective drug screening and development of treatments for DLBP.

Identification of Biomarkers, Pathways, Immune Properties of Mitophagy Genes, and Prediction Models for Intervertebral Disc Degeneration.

Background: Intervertebral disc degeneration (IDD) is the leading cause of low back pain (LBP). The mechanism of IDD development and progression is not fully understood. Peripheral biomarkers are increasingly vital non-radioactive methods in early detection and diagnosis for IDD. Nevertheless, less attention has been paid to the role of mitophagy genes in the progress of IDD. This study aimed to identify the mitophagy disease-causing genes in the process of IDD and mitophagy diagnostic biomarkers for IDD. Methods: Mitophagy-related differentially expressed genes (MRDEGs) related to IDD were investigated by analyzing the microarray datasets of IDD cases from GEO, PathCards and Molecular Signatures Databases. We used R software, WGCNA, PPI, mRNA-miRNA, mRNA-TF, GO, KEGG, GSEA, GSVA and Cytoscape to analyze and visualize the data. We further used ssGSEA for immunoinfiltration analysis to obtain different immune cell infiltration. LASSO model was developed to screen for genes that met the diagnostic gene model requirements. Finally, qRT-PCR, Western blotting and HE were used to verify hub genes and their expression from clinical IDD samples. Results: We identified 14 MRDEGs and 12 hub genes. GO, KEGG, GSEA and GSVA analyses demonstrated that hub genes were critical for the development of IDD. LASSO diagnostic model consisted of six hub genes, among which SQSTM1, ATG7 and OPTN were significantly different between the two IDD disease subtypes. At the same time, SQSTM1 also had a high correlation with immune characteristic subtypes. The results of qRT-PCR and Western blotting also indicated that these genes were significantly differentially expressed in nucleus pulposus cells (NPCs) of the IDD group. Conclusion: We explored an association between MRDEGs-associated signature in IDD and validated that hub genes like SQSTM1 might serve as biomarkers for diagnostic and therapeutic targets for IDD. Meanwhile, this study can provide new insights into the functional characteristics and mechanism of mitophagy in the development of IDD.

LncRNA XIST facilitates hypertrophy of ligamentum flavum by activating VEGFA-mediated autophagy through sponging miR-302b-3p.

BACKGROUND: Increasing evidences have shown that long non-coding RNAs (lncRNAs) display crucial regulatory roles in the occurrence and development of numerous diseases. However, the function and underlying mechanisms of lncRNAs in hypertrophy of ligamentum flavum (HLF) have not been report. METHODS: The integrated analysis of lncRNAs sequencing, bioinformatics analysis and real-time quantitative PCR were used to identify the key lncRNAs involved in HLF progression. Gain- and loss-function experiments were used to explore the functions of lncRNA X inactive specific transcript (XIST) in HLF. Mechanistically, bioinformatics binding site analysis, RNA pull-down, dual-luciferase reporter assay, and rescue experiments were utilized to investigate the mechanism by which XIST acts as a molecular sponge of miR-302b-3p to regulate VEGFA-mediated autophagy. RESULTS: We identified that XIST was outstandingly upregulated in HLF tissues and cells. Moreover, the up-regulation of XIST strongly correlated with the thinness and fibrosis degree of LF in LSCS patients. Functionally, knockdown of XIST drastically inhibited proliferation, anti-apoptosis, fibrosis and autophagy of HLF cells in vitro and suppressed hypertrophy and fibrosis of LF tissues in vivo. Intestinally, we uncovered that overexpression of XIST significantly promoted proliferation, anti-apoptosis and fibrosis ability of HLF cells by activating autophagy. Mechanistic studies illustrated that XIST directly medullated the VEGFA-mediated autophagy through sponging miR-302b-3p, thereby enhancing the development and progression of HLF. CONCLUSION: Our findings highlighted that the XIST/miR-302b-3p/VEGFA-mediated autophagy axis is involved in development and progression of HLF. At the same time, this study will complement the blank of lncRNA expression profiles in HLF, which laid the foundation for further exploration of the relationship between lncRNAs and HLF in the future.

ACSM5 inhibits ligamentum flavum hypertrophy by regulating lipid accumulation mediated by FABP4/PPAR signaling pathway.

BACKGROUND: Ligamentum flavum (LF) hypertrophy is the main cause of lumbar spinal canal stenosis (LSCS). Previous studies have shown that LF hypertrophy tissue exhibits abnormal lipid accumulation, but the regulatory mechanism remains unclear. The objective of this study was to explore the function and potential mechanism of ACSM5 in LF lipid accumulation. METHODS: To assess the ACSM5 expression levels, lipid accumulation and triglyceride (TG) level in LF hypertrophy and normal tissue, we utilized RT-qPCR, western blot, oil red O staining, and TG assay kit. The pearson correlation coefficient assay was used to analyze the correlation between ACSM5 levels and lipid accumulation or TG levels in LF hypertrophy tissue. The role of ACSM5 in free fatty acids (FFA)-induced lipid accumulation in LF cells was assessed in vitro, and the role of ACSM5 in LF hypertrophy in mice was verified in vivo. To investigate the underlying mechanisms of ACSM5 regulating lipid accumulation in LF, we conducted the mRNA sequencing, bioinformatics analysis, and rescue experiments. RESULTS: In this study, we found that ACSM5, which was significantly down-regulated in LF tissues, correlated with lipid accumulation. In vitro cell experiments demonstrated that overexpression of ACSM5 significantly inhibited FFA-induced lipid accumulation and fibrosis in LF cells. In vivo animal experiments further confirmed that overexpression of ACSM5 inhibited LF thickening, lipid accumulation, and fibrosis. Mechanistically, ACSM5 inhibited lipid accumulation of LF cells by inhibiting FABP4-mediated PPARγ signaling pathway, thereby improving hypertrophy and fibrosis of LF. CONCLUSIONS: our findings elucidated the important role of ACSM5 in the regulation of LF lipid accumulation and provide insight into potential therapeutic interventions for the treatment of LF hypertrophy. This study further suggested that therapeutic strategies targeting lipid deposition may be an effective potential approach to treat LF hypertrophy-induced LSCS.

Aberrant lncRNA-mRNA expression profile and function networks during the adipogenesis of mesenchymal stem cells from patients with ankylosing spondylitis.

Objective: We have already demonstrated that mesenchymal stem cells from patients with ankylosing spondylitis (ASMSCs) exhibited greater adipogenic differentiation potential than those from healthy donors (HDMSCs). Here, we further investigated the expression profile of long noncoding RNA (lncRNA) and mRNA, aiming to explore the underlying mechanism of abnormal adipogenic differentiation in ASMSCs. Methods: HDMSCs and ASMSCs were separately isolated and induced with adipogenic differentiation medium for 10 days. Thereafter, lncRNAs and mRNAs that were differentially expressed (DE) between HDMSCs and ASMSCs were identified via high-throughput sequencing and confirmed by quantitative real-time PCR (qRT-PCR) assays. Then, the DE genes were annotated and enriched by GO analysis. In addition, protein interaction network was constructed to evaluate the interactions between DE mRNAs and to find hub nodes and study cliques. Besides, co-expression network analysis was carried out to assess the co-expressions between DE mRNA and DE lncRNAs, and competing endogenous RNA (ceRNA) network analysis were conducted to predict the relationships among lncRNAs, mRNAs and miRNAs. The signaling pathways based on the DE genes and the predicted DE genes were enriched by KEGG analysis. Results: A total of 263 DE lncRNAs and 1376 DE mRNAs were found during adipogenesis in ASMSCs. qRT-PCR indicated that the expression of the top 20 mRNAs and the top 10 lncRNAs was consistent with the high-throughput sequencing data. Several lncRNAs (NR_125386.1, NR_046473.1 and NR_038937.1) and their target genes (SPN and OR1AIP2), together with the significantly co-expressed pairs of DE lncRNAs and DE mRNAs (SLC38A5-ENST00000429588.1, TMEM61-ENST00000400755.3 and C5orf46-ENST00000512300.1), were closely related to the enhanced adipogenesis of ASMSCs by modulating the PPAR signaling pathway. Conclusion: Our study analyzed the expression profiles of DE lncRNAs and DE mRNAs during adipogenesis in ASMSCs and HDMSCs. Several DE lncRNAs, DE mRNAs and signaling pathways that probably participate in the aberrant adipogenesis of ASMSCs were selected for future study. These results will likely provide potential targets for our intervention on fat metaplasia and subsequent new bone formation in patients with AS in the future.

Exosomes derived from PC-3 cells suppress osteoclast differentiation by downregulating miR-148a and blocking the PI3K/AKT/mTOR pathway.

Prostate cancer is a leading malignancy in men that can also disrupt the bone tissue balance. Among all urological cancers, prostate cancer is associated with the highest rate of bone metastases, which can greatly reduce a patient's quality of life. In recent years, cell-derived exosomes, which can contain a wide range of biologically active molecules, have been reported as a novel method of communication among individual cells. However, the specific role that exosomes serve in this disease has not been fully elucidated. The prostate cancer cell line PC-3 were applied in the present study, where its exosomes were isolated to explore their potential effects on osteoclast differentiation. Exosomes are extracellular vesicles secreted by cells. The size of exosomes is 30-150 nm. They have double membrane structure and saucer-like morphology. They contain rich contents (including nucleic acid, protein and lipid) and participate in molecular transmission between cells. The combined results of tartrate-resistant acid phosphatase staining (to identify osteoclasts obtained from human peripheral blood mononuclear cells), reverse transcription-quantitative PCR and western blotting showed that PC-3-derived exosomes attenuated osteoclast differentiation by downregulating marker genes associated with osteoclastic maturation, including V-maf musculoaponeurotic fibrosarcoma oncogene homolog B, matrix metalloproteinase 9 and integrin ß3. microRNA (miR)-148a expression was also found to be downregulated in osteoclasts by PC-3-derived exosomes. In addition, the mTOR and AKT signaling pathways were blocked after exposure to these PC-3 cell-derived exosomes. Therefore, results from the present study suggest that miR-148a mimics may be a new therapeutic approach for the prevention of prostate cancer bone metastases.

Integrative analysis of genome-wide DNA methylation and single-nucleotide polymorphism identified ACSM5 as a suppressor of lumbar ligamentum flavum hypertrophy.

BACKGROUND: Hypertrophy of ligamentum flavum (HLF) is a common lumbar degeneration disease (LDD) with typical symptoms of low back pain and limb numbness owing to an abnormal pressure on spinal nerves. Previous studies revealed HLF might be caused by fibrosis, inflammatory, and other bio-pathways. However, a global analysis of HLF is needed severely. METHODS: A genome-wide DNA methylation and single-nucleotide polymorphism analysis were performed from five LDD patients with HLF and five LDD patients without HLF. Comprehensive integrated analysis was performed using bioinformatics analysis and the validated experiments including Sanger sequencing, methylation-specific PCR, qPCR and ROC analysis. Furthermore, the function of novel genes in ligamentum flavum cells (LFCs) was detected to explore the molecular mechanism in HLF through knock down experiment, overexpression experiment, CCK8 assay, apoptosis assay, and so on. RESULTS: We identified 69 SNP genes and 735 661 differentially methylated sites that were enriched in extracellular matrix, inflammatory, and cell proliferation. A comprehensive analysis demonstrated key genes in regulating the development of HLF including ACSM5. Furthermore, the hypermethylation of ACSM5 that was mediated by DNMT1 led to downregulation of ACSM5 expression, promoted the proliferation and fibrosis, and inhibited the apoptosis of LFCs. CONCLUSION: This study revealed that DNMT1/ACSM5 signaling could enhance HLF properties in vitro as a potential therapeutic strategy for HLF.

KIF21B Expression in Osteosarcoma and Its Regulatory Effect on Osteosarcoma Cell Proliferation and Apoptosis Through the PI3K/AKT Pathway.

Osteosarcoma (OS) is the most common malignancy that occurs mainly during childhood and adolescence; however, no clear molecular or biological mechanism has been identified. In this study, we aimed to explore new biomarkers for the early diagnosis, targeted treatment, and prognostic determination of osteosarcoma. We first used bioinformatics analysis to show that KIF21B can be used as a biomarker for the diagnosis and prognosis of osteosarcoma. We then examined the expression of KIF21B in human osteosarcoma tissues and cell lines using immunohistochemistry, western blotting, and qRT-PCR. It was found that KIF21B expression was significantly upregulated in osteosarcoma tissues and cell lines. After knocking down the expression of KIF21B in the osteosarcoma cell lines 143B and U2-OS, we used cell fluorescence counting, CCK-8 assays, flow cytometry, and TUNEL staining to examine the effects of KIF21B on osteosarcoma cell proliferation and apoptosis. The results demonstrated that knocking down KIF21B in 143B and U2-OS cells could increase cell apoptosis, inhibit cell proliferation, and reduce tumor formation in nude mice. Subsequently, we used gene chips and bioinformatics to analyze the differential gene expression caused by knocking down KIF21B. The results showed that KIF21B may regulate OS cell proliferation and apoptosis by targeting the PI3K/AKT pathway. We then examined the expression of PI3K/AKT- and apoptosis-related proteins using western blotting. KIF21B knockdown inhibited the PI3K pathway, downregulated Bcl-2, and upregulated Bax. Moreover, the use of PI3K/AKT pathway agonists reversed the regulatory effect of KIF21B on the apoptosis and proliferation of 143B and U2-OS cells. In conclusion, our results indicated that KIF21B plays a key role in osteosarcoma. Low KIF21B expression might indirectly increase the apoptosis and inhibit the proliferation of osteosarcoma cells through the PI3K/AKT pathway.

Controlled release of FGF-2 and BMP-2 in tissue engineered periosteum promotes bone repair in rats.

The aim of this study was to prepare chitosan-collagen (CS/COL) scaffolds that could release fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein 2 (BMP-2), and to study the effect of this scaffold on bone repair. By improving the double emulsion/solvent evaporation technique, BMP-2 was encapsulated in poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PELA) microcapsules, to the surface of which FGF-2 was attached. The CS/COL scaffold carrying the microcapsules was prepared by freeze-drying. Periosteum derived cells (PDCs) were extracted and cultured on the scaffolds to study their proliferation and differentiation on the scaffolds. In addition, the effects of the scaffolds were investigated on rats with skull defects by micro-computed tomography and histology. We successfully prepared PELA microcapsules with external adherence to FGF-2 and encapsulated with BMP-2. The CS/COL scaffolds were porous and PDCs adhered, proliferated and underwent osteogenic differentiation on the scaffolds. The sequential release of FGF-2/BMP-2 had better osteogenic efficacy than other groups. Our results suggest that CS/COL scaffolds that bind FGF-2 and BMP-2 in combination with PDCs could be a promising new strategy for tissue engineering periosteum.

Animal models for studying the etiology and treatment of low back pain.

Chronic low back pain is a major cause of disability and health care costs. Effective treatments are inadequate for many patients. Animal models are essential to further understanding of the pain mechanism and testing potential therapies. Currently, a number of preclinical models have been developed attempting to mimic aspects of clinical conditions that contribute to low back pain (LBP). This review focused on describing these animal models and the main behavioral tests for assessing pain in each model. Animal models of LBP can be divided into the following five categories: Discogenic LBP, radicular back pain, facet joint osteoarthritis back pain, muscle-induced LBP, and spontaneous occurring LBP models. These models are important not only for enhancing our knowledge of how LBP is generated, but also for the development of novel therapeutic regimens to treat LBP in patients. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1305-1312, 2018.

Pharmacological targeting of the mammalian clock reveals a novel analgesic for osteoarthritis-induced pain.

Environmental disruption of the circadian rhythm is linked with increased pain due to osteoarthritis (OA). We aimed to characterize the role of the clock gene in OA-induced pain more systemically using both genetic and pharmacological approaches. Genetically modified mice, (bmal1f/fNav1.8CreERT mice), generated by deleting the critical clock gene, bmal1, from Nav1.8 sensory neurons, were resistant to the development of mechanical hyperalgesia associated with OA induced by partial medial meniscectomy (PMM) of the knee. In wild-type mice, induction of OA by PMM surgery led to a substantial increase in BMAL1 expression in DRG neurons. Interestingly, pharmacological activation of the REV-ERB (a negative regulator of bmal1 transcription) with SR9009 resulted in reduction of BMAL1 expression, and a significant decrease in mechanical hyperalgesia associated with OA. Cartilage degeneration was also significantly reduced in mice treated with the REV-ERB agonist SR9009. Based on these data, we also assessed the effect of pharmacological activation of REV-ERB using a model of environmental circadian disruption with its associated mechanical hyperalgesia, and noted that SR9009 was an effective analgesic in this model as well. Our data clearly demonstrate that genetic disruption of the molecular clock, via deletion of bmal1 in the sensory neurons of the DRG, decreases pain in a model of OA. Furthermore, pharmacological activation of REV-ERB leading to suppression of BMAL1 expression may be an effective method for treating OA-related pain, as well as to reduce joint damage associated with this disease.

PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation.

Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine-chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine-chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.

[FGF-2/PELA/BMP-2 microcapsule scaffold promotes osteogenic differentiation of rat periosteum-derived stem cells in vitro].

OBJECTIVE: To observe the effect of a microencapsule scaffold capable of sustained release of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) in promoting the osteogenic differentiation of rat periosteum-derived stem cells (PDSCs) in vitro. METHODS: PDSCs from 4-week-old SD rats, after identification of the surface markers using flow cytometry, were induced to differentiate into osteoblast, chondroblast, and adipocyte lineages. The differentiated cells were verified by staining with Alizarin red, toluidine blue, alcian blue, oil red O and by immunofluorescence assay. FGF-2/PELA/BMP-2, FGF-2/PELA, PELA/BMP-2 and PELA microcapsules were prepared, examined for surface morphologies using scanning electron microscopy (SEM), and tested for controlled release of FGF-2 and BMP-2 using ELISA. The third passage of PDSCs were cultured in the presence of the aqueous extracts of one of the 4 materials, and alkaline phosphatase (AKP) activity in the culture media was detected at 7 and 14 days of culture; the expression levels of osteogenesis-related genes were quantified with quantitative real-time PCR (qRT-PCR). The osteogenic differentiation ability of the PDSCs cultured with the extracts was compared. RESULTS: The PDSCs, which expressed mesenchymal stem cell surface markers, were shown to have osteogenic, chondrogenic and adipogenic differentiation potentials. The cells cultured with the extract of FGF-2/PELA/BMP-2 microcapsules showed the highest AKP activity at 7 and 14 days of culture, and their expression levels of OCN and RunX-2 mRNA were the highest among the 4 groups; RunX-2 expression reached its peak level on day 14, and OCN mRNA expression level increased progressively as the culture time extended. CONCLUSION: FGF-2/PELA/BMP-2 biomimetic controlled release microcapsules preserve the cytokine activities and are capable of promoting the osteogenic differentiation of rat PDSCs.

[Analysis of risk factors of nonspecific low back pain in a community population: a case-control study].

OBJECTIVE: To analyze the risk factors of nonspecific low back pain in community populations. METHODS: Two community populations were investigated using questionnaires in this case-control study. The questionnaire was designed to collect data including age, gender, body weight, marriage, education, income, occupation, labor intensity, smoking, alcohol drinking and social mental status. The subjects with low back pain constituted the case group and those without low back pain served as the control group, and the data was analyzed by a Logistic regression model. RESULTS: A total of 1747 community residents participated in this survey, among whom 398 subjects had low back pain and 1126 subjects without low back pain were selected as the control group. Of all the latent risk factors of low back pain in Logistic regression model, gender was the most relevant factor (OR=3.5522) followed by education (OR=1.958), labor intensity (OR=1.956), marital status (OR=1.612), vibration source exposure (OR=1.491), BMI (OR=1.127) and age (OR=1.060). CONCLUSION: Gender, education, labor intensity, marriage, vibration source exposure and BMI are risk factors of nonspecific low back pain in community populations, and exercises and mental status can be protective factors against low back pain.