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Symptomatic degenerative disc disease (DDD) is considered the leading cause of chronic lower back pain (LBP). As one of the main features of intervertebral disc degeneration (IDD), vascular ingrowth plays a crucial role in the progression of LBP. Stromal cell‑derived factor 1 (SDF1) and its receptor C‑X‑C receptor 4 (CXCR4) were reported to be overexpressed in the degenerated intervertebral discs, suggesting that they may be involved in the pathogenesis of IDD. Moreover, SDF1 has been identified to induce neovascularization in rheumatoid arthritis disease. However, the roles of the SDF1/CXCR4 axis in the neovascularization of IDD remain unclear. Therefore, the objective of the present study was to elucidate whether the SDF1/CXCR4 axis takes part in neovascularization in degenerated intervertebral discs and its underlying mechanisms. Adenovirus infection was used to upregulate SDF1 expression in primary nucleus pulposus cells (NPCs). The effects of SDF1 on the proliferation and angiogenesis of vascular endothelial cells (VECs) were assessed by Cell Counting Kit‑8 and tube formation assays after VECs were treated with the supernatants derived from SDF1 overexpressed or not treated NPCs. Transwell chambers using the supernatants from NPCs as chemokines were applied to assess VEC migration and invasion. AMD3100, MK‑2206 and SF1670 were used to antagonize CXCR4, AKT serine/threonine kinase 1 (AKT) and phosphatase and tensin homolog (PTEN) in VECs. The results revealed that SDF1 overexpression significantly increased the ratio of phosphorylated AKT to AKT and decreased PTEN expression in NPCs, as well as enhanced the proliferation, migration, invasion and angiogenesis abilities of VECs. However, these effects induced by SDF1 overexpression in NPCs were all reversed when VECs were pretreated with AMD3100 or MK‑2206, whereas enhanced by SF1670 treatment. Collectively, the present study indicated that enhancement of the SDF1/CXCR4 axis in NPCs can significantly accelerate angiogenesis by regulating the PTEN/phosphatidylinositol‑3‑kinase/AKT pathway.

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.

Degenerative disc disease (DDD) is the main cause of low back pain, and the ingrowth of new blood vessels is one of its pathological features. The stromal cell-derived factor 1 (SDF1)/CXCR7 signaling axis plays a role in these physiological and pathological activities. The aims of this study were to explore whether this signaling axis participates in the angiogenesis of degenerated intervertebral discs (IVDs) and to define its underlying mechanism. In this study, we cocultured human nucleus pulposus cells (NPCs) and vascular endothelial cells (VECs) and regulated the expression of SDF1/CXCR7 to investigate the effect of VEC angiogenesis by NPCs. The results revealed that angiogenesis was enhanced with increased SDF1 and that angiogenesis was weakened with the inhibition of CXCR7. We found that PI3K/AKT was involved in the downstream pathway in the coculture. VEC angiogenesis induction by NPCs was enhanced with an increase in pAKT or a decrease in PTEN. We conclude that the SDF1/CXCR7 signaling axis plays a role in the angiogenesis of degenerated IVD through the PI3K/AKT pathway.

Recent evidence suggests that Propionibacterium acnes (P. acnes) is a novel pathogenic factor promoting intervertebral disc degeneration (IVDD), however, whose mechanism remains unclear. A key component of inflammatory responses to P. acnes appears to be interleukin (IL)-1β, which has been proved to be high expression in degenerative nucleus pulposus cells (NPCs). This study aimed to explore the inflammatory mechanism driving the host response to P. acnes infection in IVDD. Our data demonstrated that the number of nod-like receptor protein 3 (NLRP3)-positive cells was significantly increased in the P. acnes-infected nucleus pulposus (NP) tissue. Meanwhile, the up-regulated expressions of NLRP3, caspase-1, caspase-5, IL-1β, IL-18, Gasdermin D (GSDMD) were observed in NPCs after co-culturing with P. acnes, which suggested NPCs pyroptosis activation induced by P. acnes. To further investigate the underlying mechanisms, NLRP3 inflammasome inhibitor MCC950 and thioredoxin binding protein (TXNIP)-siRNA were used. With the addition of MCC950 to NPCs co-cultured with P. acnes in vitro, the secretions of mature IL-1β and IL-18 were reduced. Moreover, these MCC950-mediated effects were repeated by siRNA-transfected TXNIP knockdown. These results implied P. acnes activated inflammatory response by the TXNIP–NLRP3 pathway. To further reveal the anti-degeneration role of MCC950 in vivo, MCC950 was injected into the rabbit IVDD models infected by P. acnes. The MRI and histological detection provided more solid evidence that MCC950 treatment effectively retarded the degenerative process of the intervertebral discs in vivo. In summary, these results suggest that P. acnes-induced NPCs pyroptosis activation via the NLRP3-dependent pathway is likely responsible for the inflammatory pathology of IVDD. MCC950 can alleviate inflammatory injury and NPCs pyroptosis under P. acnes infection and may delay the progression of disc degeneration, which provides a new direction for the treatment of IVDD.

Backgrounds Adipose derived stem cells (ADSCs) could undergo osteogenesis via focal adhesion kinase (FAK) and bone morphogenetic protein (BMP) 9 signals, both of which could affect Wnt-β-catenin signal, a signal pathway closely related to ADSCs osteogenesis. It’s still enigma whether FAK and BMP-9 contribute to osteogenesis. Here, we examined the effect of FAK on BMP9-inducedosteogenic differentiation, unveiled the possible molecular mechanism underling this process. Methods In the present study, ADSCs were isolated and purified, and cells of passage 3 underwent virus mediated transfection to prepare ADSCs with stable FAK shRNA expression. Cell viability and migration were detected by MTT and transwell assay, respectively. Expression of osteogenic gene, phosphorylation of FAK and GSK were detected by western blot. Osteogenic potential was evaluated by activity of alkaline phosphatase (ALP) and calcium deposition by ALP staining and Alizarin Red S staining. Results BMP-9 administration promoted ADSCs osteogenesis. Knocking down FAK attenuated this process, inhibited osteogenic proteins expression through Wnt-β-catenin signal. BMP-9 also triggered ADSCs proliferation and migration, and shFAK antagonized such effects too. Although Wnt signal is affected by FAK shRNA, Smad signal remains intact in ADSCs with shFAK. Conclusion FAK and BMP-9 could cross talk on Wnt signal pathway and promote ADSCs osteogenesis. FAK could participate in BMP-9 induced ADSCs osteogenesis via Wnt signal pathway other than Smads signals (see in graph).

Schisandrin B is an active monomer of the Chinese magnolia vine (Schisandra chinensis) that can reduce transaminase activity in liver cells, inhibit lipid peroxidation, enhance antioxidant status, has protective effects in the liver and has antitumor effects. The present study investigated the potential protective effects of schisandrin B on the p53 signaling pathway in attenuating the inflammatory response, oxidative stress and apoptosis induced by traumatic spinal cord injury (TSCI) in adult rats. Behavioral examination, inclined plate test and spinal cord water content were used to evaluate the protective effect of schisandrin B in TSCI rats. The expression levels of superoxide dismutase (SOD), malondialdehyde (MDA), nuclear factor (NF)‑κB subunit p65 and tumor necrosis factor (TNF)‑α were examined using ELISA kits. Western blot analysis was performed to analyze the protein expression of caspase‑3 and phosphorylated (p)‑p53 in TSCI rats. In the present study, schisandrin B improved behavioral examination results and the maximum angle of inclined plate test, and inhibited spinal cord water content in rats with TSCI. Notably, schisandrin B reduced the activation of traumatic injury‑associated pathways, including SOD, MDA, NF‑κB p65 and TNF‑α, in TSCI rats. In addition, schisandrin B suppressed the TSCI‑induced expression of caspase‑3 and p‑p53 in TSCI rats. These results indicated that schisandrin B may attenuate the inflammatory response, oxidative stress and apoptosis in TSCI rats by inhibiting the p53 signaling pathway in adult rats.

Background: MicroRNAs (miRNAs) are involved in the regulation of osteogenic differentiation and chondrification in vivo. The purpose of the present study was to explore the potential mechanism of miR-128 in osteoporosis (OP). Methods: Quantitative real-time PCR (qRT-PCR) was used to determine the expression of miR-128 in femoral neck trabecular bones of OP patients (n=40) and non-OP patients (n=40). C2C12 cells were transfected with miR-128 mimic or inhibitor to determine the effect of miR-128 on osteoblastic differentiation of C2C12 cells. Bioinformatics and luciferase reporter genes were used to determine the molecular mechanism of miR-128 in osteoblastic differentiation of C2C12 cells. Results: The qRT-PCR results showed that the expression level of miR-128 in bone samples of OP patients was significantly higher than that of non-OP patients, while miR-128 was significantly down-regulated during the osteogenic differentiation of C2C12 cells. In addition, the results showed that overexpression of miR-128 significantly inhibited the mRNA and protein expression levels of osteocalcin (OC), alkaline phosphatase (ALP) and collagen I type-α1 (COL1A1) in C2C12 cells, while miR-128 inhibitor could reverse this effect. Bioinformatics analysis and dual-luciferase reporter assay found that silencing information regulatory protein 6 (SIRT6) was a direct target of miR-128. The qRT-PCR and Western Blot results found that miR-128 significantly down-regulated the mRNA and protein expressions of SIRT6. Furthermore, silencing SIRT6 significantly inhibited the promoting effect of the miR-128 inhibitor on the expression of osteoblast markers. Conclusion: The above results confirmed that miR-128 inhibited osteoblast differentiation in OP by down-regulating SIRT6 expression, thus accelerating the development of OP.

To evaluate the results of percutaneous endoscopic transforaminal diskectomy (PETD) in comparison with percutaneous endoscopic interlaminar diskectomy (PEID) for axillar herniation at L5-S1.From January 2017 to March 2018, 80 patients admitted with axillar herniation at L5-S1 were randomly recruited into 2 groups: 40 cases in the PETD group and 40 in the PEID group. Each group separately underwent PETD or PEID. Patient sex, age, body mass index, axillar herniation size, number of C-arm fluoroscopies, operation time, postoperative bed time, complications, and clinical effect were compared. Both groups were followed-up using the Oswestry Disability Index (ODI), visual analog scale (VAS), and Macnab criteria.Except for 1 case in the PETD group that switched to the PEID group, the patients completed the study as expected. All patients were followed-up. Preoperative demographics were not significantly different (P0.05) between the 2 groups. The mean number of C-arm fluoroscopies (12.44 ± 3.21) and the operation time (66.49 ± 16.29 minutes) of the PETD group were significantly improved compared with the PEID group (number of fluoroscopies: 3.41 ± 0.81, P0.001; operation time: 53.56 ± 10.82 minutes, P0.001), but the postoperative bed rest time and complication rate were not (P0.05). The postoperative ODI and VAS scores were obviously improved in both groups when compared with preoperation (P0.001). There were no significant differences between the 2 groups in the Macnab criteria or VAS and ODI scores at the same time point (P0.05).For axillar herniation at L5-S1, PEID can ignore the anatomic obstruction with advantages including a shorter operation time and less intraoperative radiation exposure. PETD has a clinical effect similar to that of PEID, but the process of it is more dangerous and harder than PEID.

Polymethyl methacrylate (PMMA) bone cement has been commonly used for percutaneous injection into collapsed vertebral bodies due to malignant tumor. The purpose of this study was to investigate the possible mechanisms of PMMA’s cytotoxcity on primary cultured spinal metastastic cells (SMCs) in vitro. PMMA specimens were prepared in standard discs made of dough and polymerization stages, and the eluates were prepared following the ISO standard. Primary SMCs were obtained and isolated from 7 patients with spinal metastatic tumors undergoing vertebroplasty. Primary cultured SMCs were treated with PMMA specimens of different stages for 24 h, or co-cultured with extracted medium for successive 3 days. The temperatures in two locations from cement discs were recorded by K-type thermocouples. Furthermore, cell proliferation, apoptosis and cycles were determined by MTT and flow cytometry, respectively. The modulation of apoptotic proteins (bcl-2, bax, caspase-3, caspase-8, Fas and PARP) and cell cycle proteins (cyclin D1, P21 and P27) were analyzed by western blot and real time PCR. The results of this study demonstrated that PMMA treatment was able to suppress SMCs proliferation, induce cell apoptosis and inhibit cell cycle arrest when compared to the control group in vitro (P

Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

RSK2 (90 kDa ribosomal S6 kinase) is a downstream effector of the Ras/ERK (extracellular signal-regulated kinase) signaling pathway that has major functions in cell biological activities, including regulating nuclear signaling, cell cycle progression, cell proliferation, cell growth, protein synthesis, cell migration and cell survival, and is expressed in most types of human malignant tumors, including lung cancer, prostate and breast tumors, skin cancer and osteosarcomas (OS). RSK2 was found to be essential for osteosarcoma formation. To investigate whether RSK2 is expressed at high levels in human osteosarcome tissues and whether its expression is correlated with the aggressive biological behavior of osteosarcoma cell line (OCLs), we assessed the association between RSK2 expression and OS cell progression, as well as the effects of RSK2 inhibition on the biological activities of osteosarcoma cells. We performed immunohistochemistry to analyze the expression of RSK2 in specimens from 30 humans with osteosarcoma, and 15 normal tissues. RSK2 gene expression levels in 30 specimens with osteosarcoma were significantly higher than those of normal tissues. We performed RNA interference on three OCLs to evaluate cell apoptosis, cell growth, cell proliferation, cell motility, chemosensitivity and oncogenicity. After transfection with RSK2 shRNA, increased cell apoptosis, cell growth inhibition, cell cycle progression, weaker cell proliferation, cell migration and weaker tumor formation were observed in all OCLs. These results suggested that RSK2 expression may mediate the biological activities of OS cells and RSK2 may be an effective therapeutic target for the treatment of osteosarcomas. The AKT/mTOR, MAPK/ERK/c-Fos and Bcl2/Bax pathways were analysed to clarify the mechanisms involved.

STUDY DESIGN In vitro experimental study. OBJECTIVE To investigate the effect of low-intensity pulsed ultrasound (LIPUS) on the extracellular matrix (ECM) synthesis of degenerative human nucleus pulposus cells and explore the molecular mechanism. SUMMARY OF BACKGROUND DATA LIPUS has been used successfully for bone fracture healing and been proved to be effective in stimulating ECM metabolism in animal intervertebral disc cells. However, whether LIPUS also exerts an anabolic effect on degenerative human nucleus pulposus cells and the possible molecular mechanism is still unclear. METHODS The degenerative human nucleus pulposus cells were cultured in calcium alginate beads. In the LIPUS group, cells were exposed to an average temporal intensity of 30 mW/cm2 and a frequency of 1.5 MHz of LIPUS 20 minutes daily for 1 week. The control group was cultured in the same way but without LIPUS stimulation. The LY294002 group was stimulated by LIPUS and treated with LY294002 simultaneously. The expression of aggrecan, collagen-II, Sox9, tissue inhibitor of metalloproteinase-,1 and matrix metalloproteinase-3 were evaluated by Enzyme-Linked Immunosorbent Assay, Western blot or RT-PCR. Expression of signaling proteins involved in FAK/PI3K/Akt pathway was studied by Western blot analysis. RESULTS LIPUS significantly upregulated expression of aggrecan, collagen-II, Sox9, and tissue inhibitor of metalloproteinase-1 compared with control group, but inhibited secretion of matrix metalloproteinase-3. The study further demonstrated that the upregulation of aggrecan, collagen-II, and Sox9 was related to the activation of focal adhesion kinase (FAK)//PI3K/Akt pathway caused by LIPUS. Moreover, inhibition of PI3K/Akt significantly suppressed the special biological effect activated by LIPUS. CONCLUSION LIPUS promotes the ECM synthesis of degenerative human nucleus pulposus cells through activation of FAK/PI3K/Akt pathway. LEVEL OF EVIDENCE N/A.

Background: Low back pain (LBP), one of the most prominent problems worldwide, lacks effective disease-modifying medical therapy. Intervertebral disc degeneration (IVDD) is a major cause of LBP, and proinflammatory cytokines are the key factors involved in the development of IVDD-induced back pain. Sirtuin 1 (SIRT1) is implicated in the molecular control of aging and immune responses in various diseases; however, its effect on IVDD is not clearly understood. Objective: To investigate the effects of SIRT1 on proinflammatory stress and signal transduction pathways induced by interleukin-1β (IL-1β) in human degenerative nucleus pulposus (NP) cells. Study Design: Research study. Setting: Chongqing Key Laboratory of Ophthalmology. Methods: Anti-apoptotic and anti-catabolic effects of SIRT1 on IL-1β were investigated using a three-dimensional cell culture model of prestimulated human NP cells transfected with a lentiviral vector overexpressing SIRT1 or a small-interfering RNA (siRNA) against the gene encoding SIRT1. In addition, molecular mechanisms underlying the association of SIRT1 with Toll-like receptor-2 (TLR2) and nuclear factor kappa B (NF-κB) were investigated. Results: Our results indicated that decreased SIRT1 expression was associated with IVDD. Direct regulation of SIRT1 expression did not affect the synthesis of extracellular matrix (ECM). However, SIRT1 overexpression mediated by the lentiviral vector suppressed IL-1β-induced ECM degradation and cell apoptosis. In contrast, knockdown of the gene encoding SIRT1 by the siRNA increased MMP expression and cell apoptosis induced by IL-1β. Furthermore, SIRT1 deacetylated RelA/p65 to inhibit the nuclear translocation of NF-κB, thus inhibiting inflammation. On the other hand, IL-1β downregulated SIRT1 expression by activating TLR2. However, inhibition of TLR2 expression by an siRNA did not inhibit IL-1β-induced nuclear translocation of NF-κB. Limitations: Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of SIRT1 on IVDD. Conclusions: Our results indicated that SIRT1 exerted anti-inflammatory effects against IL-1βmediated degeneration of NP cells through the TLR2/SIRT1/NF-κB pathway, suggesting that it could be used as a potential candidate for treating IVDD-mediated back pain. Key words: SIRT1, TLR2, NF-κB, intervertebral disc degeneration

Bone morphogenetic protein 9 (BMP9) is a potent inducer of osteogenic differentiation of mesenchymal stem cells. The Wnt antagonist Dickkopf-1 (Dkk1) is involved in skeletal development and bone remodeling. Here, we investigated the role of Dkk1 in BMP9-induced osteogenic differentiation of MSCs. We found that overexpression of BMP9 induced Dkk1 expression in a dose-dependent manner, which was reduced by the P38 inhibitor SB203580 but not the ERK inhibitor PD98059. Moreover, Dkk1 dramatically decreased not only BMP9-induced alkaline phosphatase (ALP) activity but also the expression of osteocalcin (OCN) and osteopontin (OPN) and matrix mineralization of C3H10T1/2 cells. Furthermore, exogenous Dkk1 expression inhibited Wnt/β-catenin signaling induced by BMP9. Our findings indicate that Dkk1 negatively regulates BMP9-induced osteogenic differentiation through inhibition of the Wnt/β-catenin pathway and it could be used to optimize the therapeutic use of BMP9 and for bone tissue engineering. [BMB Reports 2016; 49(3): 179-184].

Intervertebral disc degeneration (IDD) is a main cause of diseases such as discogenic low back pain, cervical and lumbar disc herniation, degenerative spinal stenosis, and lumbar spondylolisthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2), an important transcription factor, regulates antioxidant genes and induces cellular defense mechanisms against oxidative stress. In this study, the protective effect of plant antioxidant lycopene on nucleus pulposus cells (NPCs) under oxidative stress was investigated. The results indicated that Nrf2 expression decreased in degenerated NPCs. We further found that lycopene was protective in NP tissue under oxidative stress and alleviated oxidative stress-induced apoptosis of degenerative human NPCs via Nrf2. The results also showed that lycopene reduced H2O2-induced decomposition of cartilage extracellular matrix in NPCs. In conclusion, our findings suggested that lycopene may alleviate disc degeneration under oxidative stress through the Nrf2 signaling pathway.

Extracellular matrix (ECM) anabolism and catabolism imbalance is key feature of chondrocyte and intervertebral disc nucleus pulposus (NP) cell degeneration. The role of LIF as a multifunctional cytokine in the ECM metabolism of chondrocytes is controversial, but no relevant research in the ECM metabolism of NP cells. This study aimed to explore the biofunction and related mechanisms of LIF in the degenerative NP cells. We obtained an increase in the expression of LIF in the human degenerated NP specimens. The addition of recombinant human leukemia inhibitory factor (rhLIF) to the degenerated NP cells cultured in vitro was found to stimulate the synthesis of ECM, and rhLIF could activate the ERK1/2 signaling pathway. However, coculture with PD98059, a signal inhibitor of ERK1/2, blocked the effect of rhLIF on the synthesis of ECM. To furtherly clarify the role of LIF, we carried out animal experiments and found that rhLIF treatment could successfully delay the degree of degeneration of the intervertebral disc in a rabbit model; but with the addition of PD98059, the function of rhLIF for degeneration protection disappeared. In summary, this study demonstrates that LIF plays a role in promoting ECM synthesis in the degenerated NP cells as a protective role in intervertebral disc degeneration (IDD), which is related to the activation of ERK1/2 signaling pathway.

Intervertebral disc degeneration (IDD) is closely related with aging, whereas mitochondrial dysfunction is a common feature of aging in which results cell senescence. Phosphatase and tensin homolog (PTEN)-induced putative kinase protein 1 (PINK1) is a mitochondrial-targeted serine/threonine kinase, which plays a protective role against mitochondrial dysfunction with mitochondrial quality control by activating PINK1/Parkin mediated mitophagy. This study aimed to investigate the protective role of PINK1 against mitochondrial dysfunction and human nucleus pulposus cell (NPC) senescence. We found that mitochondrial dysfunction and NPC senescence could be induced under sublethal oxidative stress by 150 μM H2O2. Moreover, down-regulation of PINK1 tended to aggravate NPC senescence under oxidative stress. Therefore, mitophagy was evaluated in NPCs to further reveal the underlying mechanism. Results showed that sublethal oxidative stress induced mitochondria dysfunction and mitophagy in NPCs. Furthermore, depletion of PINK1 utilizing short hairpin RNA targeting PINK1 (PINK1-shRNA) impaired mitophagy, and exasperated NPC senescence under oxidative stress. In summary, these results suggested that PINK1 played as a protective role in clearance of damaged mitochondrial and alleviating cell senescence under oxidative stress, whose mechanism is associated with regulating mitophagy. These findings may provide a better understanding in pathomechanism of IDD and potential therapeutic approaches for IDD treatment.

Aim: To improve nucleus pulposus cell-targeted therapy for intervertebral disc degeneration (IDD) by fabricating a novel kind of ultrasound (US)-mediated poly(lactic-co-glycolic acid) nanobubbles (NBs) as a means of targeted drug delivery. Materials & methods: The resveratrol (RES)-embedded NBs were synthesized using a double-emulsion method. The active NP cell-targeting biomarker CDH2 antibody (AbCDH2) was further conjugated to the NBs using a carbodiimide method. Then, this RES/AbCDH2 NBs were examined by physical properties, specifc cell-targeting ability, anticatabolism effect in vitro and in vivo. Results: RES/AbCDH2 NBs exhibited high RES-loading efficiency, and US triggered accelerated RES release. Furthermore, RES/AbCDH2 NB treatment exhibited excellent anticatabolic ability in vitro; and in an IDD rabbit model, US-mediated RES/AbCDH2 NB injection effectively retarded the degenerative process of the intervertebral disc in vivo. Conclusion: The combination of US irradiation and drug delivery through RES/AbCDH2 NBs can be considered as a novel treatment option for IDD.

The present study was performed to compare the clinical efficacy of lateral plate and lateral and medial double-plating fixation of distal femoral fractures and explore the indication of lateral and medial double-plating fixation of the distal femoral fractures. From March 2006 to April 2014, 48 and 12 cases of distal femoral fractures were treated with lateral plate (single plate) and lateral and medial plates (double plates), respectively. During the surgery, after setting the lateral plate for the distal femoral fractures, if the varus stress test of the knee was positive and the lateral collateral ligament rupture was excluded, lateral and medial double-plating fixation was used for the stability of the fragments. All the patients were followed up at an average period of 15.9 months. The average operation time, the intraoperative hemorrhage and the fracture union time of the two groups were compared. One year after operation, knee function was evaluated by the Kolmert’s standard. There was no significant difference in the average operation time, intraoperative hemorrhage, fracture healing time and excellent and good rates of postoperative knee function between two groups. Positive Varus stress test during operation can be an indication for lateral and medial double-plating fixation of distal femoral fractures.

Various gene delivery systems have been widely studied for the acute spinal cord injury (SCI) treatment. In the present study, a novel type of brain-derived neurotrophic factor (BDNF)-loaded cationic nanobubbles (CNBs) conjugated with MAP-2 antibody (mAbMAP-2/BDNF/CNBs) was prepared to provide low-intensity focused ultrasound (LIFU)-targeted gene therapy. In vitro experiments, the ultrasound-targeted tranfection to BDNF overexpressioin in neurons and efficiently inhibition neuronal apoptosis have been demonstrated, and the elaborately designed mAbMAP-2/BDNF/CNBs can specifically target to the neurons. Furthermore, in a acute SCI rat model, LIFU-mediated mAbMAP-2/BDNF/CNBs transfection significantly increased BDNF expression, attenuated histological injury, decreased neurons loss, inhibited neuronal apoptosis in injured spinal cords, and increased BBB scores in SCI rats. LIFU-mediated mAbMAP-2/BDNF/CNBs destruction significantly increase transfection efficiency of BDNF gene both in vitro and in vivo, and has a significant neuroprotective effect on the injured spinal cord. Therefore, the combination of LIFU irradiation and gene therapy through mAbMAP-2/BDNF/CNBs can be considered as a novel non-invasive and targeted treatment for gene therapy of SCI.

// Peng Wang 1 , Lizhu Jiang 2 , Nian Zhou 1 , Hao Zhou 1 , Huzhe Liu 1 , Wenrui Zhao 1 , Hanxiang Zhang 1 , Xiang Zhang 1 and Zhenming Hu 1 1 Department of Orthopedic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China 2 Department of Otorhinolaryngology Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China Correspondence to: Zhenming Hu, email: 182315140@qq.com Keywords: resveratrol; spinal cord injury; autophagy flux; apoptosis; LKB1/AMPK/mTOR/p70s6k pathway Received: June 13, 2017 Accepted: November 08, 2017 Published: January 03, 2018 ABSTRACT Resveratrol is known to improve functional recovery after spinal cord injury, but the exact mechanism involved is yet unclear. The aim of this study was to clarify whether resveratrol can exert neuroprotective effects via activating neuronal autophagic flux, in view of the underlying role of the autophagic flux mediated by resveratrol on neuronal apoptosis after spinal cord injury, and identify the role of the liver kinase B1(LKB1)/adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/ p70 ribosomal protein S6 kinase (p70s6k) signal pathway in the autophagic flux mediated by resveratrol. The results obtained strongly indicate that resveratrol improved functional recovery in Sprague–Dawley rats after acute spinal cord injury, preserved their motor neurons, alleviated the neuronal apoptosis, and ameliorated neuronal autophagic flux. After blocking the autophagic flux, the neuroprotective effects of resveratrol were eliminated. Furthermore, it was proved that resveratrol can activate the LKB1/AMPK/mTOR/p70s6k pathway in vivo and in vitro , and the LKB1/AMPK/mTOR/p70s6k pathway plays a vital role in activating the autophagic flux mediated by resveratrol in PC12 cells. Thus, resveratrol enables to ameliorate neuronal autophagic flux via the LKB1/AMPK/mTOR/p70s6k pathway to alleviate apoptosis, and finally ameliorating functional recovery after acute SCI in SD rats.

Zhaojun Song,1 Zhigang Wang,2 Jieliang Shen,1 Shengxi Xu,1 Zhenming Hu1 1Department of Orthopedics, The First Affiliated Hospital, 2Institution of Ultrasound Imaging, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, People’s Republic of China Background: Spinal cord injuries (SCIs) can cause severe disability or death. Treatment options include surgical intervention, drug therapy, and stem cell transplantation. However, the efficacy of these methods for functional recovery remains unsatisfactory. Purpose: This study was conducted to explore the effect of ultrasound (US)-mediated destruction of poly(lactic-co-glycolic acid) (PLGA) nanobubbles (NBs) expressing nerve growth factor (NGF) (NGF/PLGA NBs) on nerve regeneration in rats following SCI. Materials and methods: Adult male Sprague Dawley rats were randomly divided into four treatment groups after Allen hit models of SCI were established. The groups were normal saline (NS) group, NGF and NBs group, NGF and US group, and NGF/PLGA NBs and US group. Histological changes after SCI were observed by hematoxylin and eosin staining. Neuron viability was determined by Nissl staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining was used to examine cell apoptosis. NGF gene and protein expressions were detected by quantitative reverse transcription polymerase chain reaction and Western blotting. Green fluorescent protein expression in the spinal cord was examined using an inverted fluorescence microscope. The recovery of neural function was determined using the Basso, Beattie, and Bresnahan test. Results: NGF therapy using US-mediated NGF/PLGA NBs destruction significantly increased NGF expression, attenuated histological injury, decreased neuron loss, inhibited neuronal apoptosis in injured spinal cords, and increased BBB scores in rats with SCI. Conclusion: US-mediated NGF/PLGA NBs destruction effectively transfects the NGF gene into target tissues and has a significant effect on the injured spinal cord. The combination of US irradiation and gene therapy through NGF/PLGA NBs holds great promise for the future of nanomedicine and the development of noninvasive treatment options for SCI and other diseases. Keywords: nanobubbles, nerve growth factor, spinal cord injury, apoptosis, gene therapy

IL-1β has been reported highly expressed in degenerative intervertebral disc, and our previous study indicated IL-1β facilitates apoptosis of human degenerative nucleus pulposus (NP) cell. However, the underlying molecular mechanism remains unclear. We here demonstrate that IL-1β played a significantly pro-apoptotic effect under serum deprivation. IL-1β decreased Bcl-2/Bax ratio and enhanced cytochrome C released from mitochondria to cytosol, which proved mitochondria-meidated apoptosis was induced. Subsequently, mitochondria damage was detected under IL-1β stimualtion. In addition, IL-1β-mediated injuried mitochondria contributes to activate autophagy. However, pretreatment with the autophagy inhibitor 3-methyladenine showed the potential in further elevating the apoptosis rate induced by IL-1β in NP cells. Our results indicated that the mitochondrial pathway was involved in IL-1β-induced apoptosis of NP cells. Meanwhile, the damaged mitochondria-induced autophagy played a protective role against apoptosis, suggesting a postive feedback mechanism under inflammatory stress.

Intervertebral disc degeneration (IVDD) is a major cause of lower back pain, and increased cell apoptosis is a key characteristic of IVDD. The present study aimed to investigate the effects and mechanism of the stromal cell-derived factor-1 (SDF-1)/C-X-C motif chemokine receptor 4 (CXCR4) axis on apoptosis in human degenerative nucleus pulposus cells (NPCs). The expression levels of SDF-1 and CXCR4 in human intervertebral discs (IVD) were determined using immunohistochemistry and western blot analysis. Apoptosis of primary cultured NPCs was quantified by Annexin V/propidium iodide staining following stimulation with SDF-1 and knockdown of CXCR4 using small interfering RNA (siRNA). The association with the nuclear factor-κB (NF-κB) signaling pathway was investigated using CXCR4-siRNA and NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), treatment. The results demonstrated that SDF-1 and its receptor, CXCR4, were upregulated in degenerative IVD samples compared with normal samples. Stimulation with SDF-1 increased the level of apoptosis in cultured NPCs, and conversely, the apoptosis level was suppressed post-transfection with CXCR4 siRNA compared with SDF-1 stimulation alone. Furthermore, SDF-1 treatment increased the level of phosphorylated NF-κB subunit P65, which was downregulated following CXCR4 siRNA and PDTC treatment. In addition, CXCR4 siRNA and PDTC inhibited the nuclear translocation of P65, which was induced by SDF-1. Taken together, SDF-1-mediated apoptosis was suppressed by NF-κB inhibition using PDTC. In conclusion, the SDF-1/CXCR4 axis promoted cell apoptosis in human degenerative NPCs via the NF-κB pathway, thus suggesting that SDF-1/CXCR signaling may be a therapeutic target for the treatment of degenerative IVD diseases.

The purpose of this article was to systematically review the clinical outcomes of microendoscopic foraminotomy compared with the traditional open cervical foraminotomy. A literature search of two databases was performed to identify investigations performed in the treatment of cervical foraminotomy with microsurgery or an open approach. Data including blood loss, surgical time, hospital stay, complications, clinical success rate, reduction of arm and neck pain, improvement of neurological function, and repeated surgery rate were summarized, calculated and compared. Results of clinical success were performed by calculattng effect indicators and standard errors based on a single rate to assess heterogeneity in the two groups. The initial literature search resulted in 713 articles, of which, 26 were determined as relevant on abstract review. An open foraminotomy approach was performed in 16 and a microsurgery approach in ten studies. The open group demonstrated minimal to moderate heterogeneity, with I 2 value of 27 %; and microsurgery group demonstrated minimal heterogeneity, with I 2 value of 1 %. Aggregated data found that patients treated by microsurgery foraminotomy have lower blood loss by 100.1 ml (open: 149.5 ml, microsurgery: 49.4 ml, n = 1257), shorter surgical time by 24.9 minutes (open 88.7 minutes, microsurgery 63.8 minutes, n = 1423),and shorter hospital stay by 3.0 days (open 4.1 days, microsurgery 1.1 days, n = 1350), compared with patients treated by open cervical foraminotomy. The pooled clinical success rate was 89.7 % [confidence interval (CI) 87.7–91.6) in the open group versus 92.5 % (CI 89.9–95.1) in the microsurgery group, with no statistical difference (p = 0.095). Overall complication rates were not statistically significant between groups (p = 0.757). The incidence of dural tears was 1.07 %( 12/1121) in patients undergoing microsurgery versus 0.27 % (2/745) for open surgery (p = 0.091). The incidence of infection was 0.54 % (6/1121) in patients undergoing microsurgery versus 0.40 % (3/745) for open surgery (p = 0.949). The incidence of root injury was 0.80 % (9/1121) in patients undergoing microsurgery versus 1.48 % (11/745) for open surgery (p = 0.166). Revision surgery occurred in 2.32 % (27/1163) in the microsurgery group versus 3.35 % (28/835) for traditional surgery, with no statistical difference (p = 0.164). Pooled reduction in visual analogue scale for the arm (VASA) was 75.0 % (CI 66.0–84.0) in the open group and 87.1 % (CI:76.7, 97.5) in the microsurgery group, with no statistical difference (p = 0.065). Pooled reduction in VAS of the neck (VASN) was 66.2 % (CI:52.2, 80.2) in the open group and 68.1 % (CI:36.4, 99.8) in the microsurgery group, with no statistical difference(p = 0.894). Pooled improvement in neurological function was 55.3 % (CI:18.6, 91.9) in the open group and 64.9 % (CI:34.6, 95.2) in the microsurgery group, with no statistical difference (p = 0.576). Although advantages of cervical microsurgery are less blood loss and shorter surgical time and hospital stay over the standard open technique, there is no significant difference in clinical success rate, complication rate, reduction of arm and neck pain and improvement of neurological function between microsurgery and open cervical foraminotomy.

Point-of-care (POC) testing offers rapid diagnostic results. However, the quantification of current methods is performed using standard curves and external references, and not direct and absolute quantification. This paper describes an integrated multiplex digital recombinase polymerase amplification (ImdRPA) microfluidic chip which combines DNA extraction, multiplex digital RPA and fluorescence detection together in one chip, creating a “sample-in-multiplex-digital-answer-out” system. Multi-layer soft lithography technology was used, with polydimethylsiloxane (PDMS) as the chip material and a glass slide as the substrate. This microfluidic chip has a six-layer structure and screw microvalve control function. The sample preparation for the chip involved magnetic bead-based nucleic acid extraction, which was completed within 15 min without any instrument dependence. The dRPA region was divided into 4 regions (3 positive detection areas and 1 negative control area) and included a total of 12 800 chambers, with each chamber being able to contain a volume of 2.7 nL. The screw valve allowed for the reaction components of each specific goal to be pre-embedded in different regions of the chambers. The reagents were passively driven into the dRPA region using vacuum-based self-priming introduction. Furthermore, we successfully demonstrated that the chip can simultaneously detect three species of pathogenic bacteria within 45 min and give digital quantitative results without the need to establish a standard curve in contaminated milk. Moreover, the detection limit of this ImdRPA microfluidic chip was found to be 10 bacterial cells for each kind of pathogen. These characteristics enhance its applicability for rapid detection of foodborne bacteria at the point-of-care (POC). We envision that the further development of this integrated chip will lead to rapid, multiplex and accurate detection of foodborne bacteria in a feasible manner.