Kuibo Zhang

Role of death receptor, mitochondrial and endoplasmic reticulum pathways in different stages of degenerative human lumbar disc

Intervertebral disc (IVD) cell apoptosis has been suggested to play an important role in promoting the degeneration process. It has been demonstrated that IVD cell apoptosis occurs through either death receptor, mitochondrial or endoplasmic reticulum (ER) pathway. Our study aimed to explore the relationship among these three pathways and grade of IVD degeneration (IVDD). IVDs were collected from patients with lumbar fracture, vertebral tumor, disc herniation or spondylolisthesis. IVDs were distinguished by MRI and histomorphological examination, cell apoptosis was detected by TUNEL staining. Biomarkers of these three apoptosis pathways were detected by RT-PCR and Western blot. Furthermore, the correlation between apoptosis pathways biomarkers and disc pathology were analyzed. Nucleus pulposus cell density decreased with degeneration process, and increased apoptotic ratio. ER pathway was predominant in mild stage of IVDD (GRP78, GADD153 upregulation and caspase-4 activation), death receptor pathway was predominant in mild and moderate stages (Fas, FasL up-regulation and caspase-8 activation) and mitochondrial pathway was predominant in moderate and severe stages (Bcl-2 down-regulation, Bax up-regulation, cytochrome-c accumulation in cytoplasm and caspase-9 activation). There were significant differences in the expressions of Fas, FasL, Bax, GADD153, cytochrome-c and cleaved caspase-8/9/3 between contained and non-contained discs. In conclusion, apoptosis occurs via these three apoptosis pathways together in IVDD. ER pathway plays a more critical role in the mild compared to moderate and severe stages, death receptor pathway in mild and moderate, and mitochondrial pathway in moderate and severe stages of IVDD. Disc cells apoptosis may progress rapidly after herniation, and may depend on the type of herniation.

The effect of screw length and bone cement augmentation on the fixation strength of iliac screws: a biomechanical study.

Study Design Comparison of the biomechanical fixation strengths offered by 3 iliac screw fixation techniques: short screw, short screw augmented with cement, and long screw. Objective Evaluate the effect of screw length and bone cement augmentation on the fixation strength of iliac screw upon fatigue loading. Summary of Background Data Iliac screws have been used in treating spinal disorders such as spinal deformity, spondylolisthesis, and sacral tumor. In clinical practices, both short screws and long screws are being used. It has been reported that short iliac screws have a higher rate of loosening. Therefore, short iliac screws are being used with bone cement augmentation to improve fixation. To date, no biomechanical study has compared the strengths of these 3 different iliac screw fixation techniques. Method Fresh, frozen human cadaveric pelvis specimens (n=18, 12 males, 6 females, average age 61 y) were used. Bone density was measured to characterize bone quality. The specimens were randomly divided into 2 groups. In group 1 (n=8), short screws of 7.0-mm diameter and 70±4 mm length (as the length of exceeding over ischial notch) and long screw of 7.0-mm diameter and 120±4 mm length were placed on either side of the pelvis (left and right). In group 2 (n=10), short iliac screws were placed after augmentation with polymethyl methacrylate bone cement on 1 side of the pelvis and long iliac screw were placed on the other side (left and right). Cyclic loading ranging from 20 to 200 N was applied to each screw at a frequency of 2 Hz up to 5000 cycles. Pullout tests were then conducted at the rate of 5 mm/min after the fatigue test, and the maximum pullout strength for each screw was recorded and analyzed. Results The maximum pullout strength of the long screw and short screw groups after fatigue conditioning were 2386±1470 and 833±681 N respectively. Significant difference was found between the 2 groups (P<0.05). The short iliac screw had a higher loosening rate. The pullout force of the short screw fixation with augmentation and the long screw fixation after cyclic loading were 2436±915 and 2529±1055 N, respectively. No significant difference was found between the 2 groups (P>0.05). Conclusions Short iliac screws are susceptible to loosening after cyclic loading. Bone cement augmentation of short screws has demonstrated a significant increase in the fixation strength of short screws to an extent similar to that of long iliac screws. Thus, short iliac screw fixation after augmentation with bone cement will be a viable clinical option for spino-pelvic reconstruction.

LIM mineralization protein-1 suppresses TNF-α induced intervertebral disc degeneration by maintaining nucleus pulposus extracellular matrix production and inhibiting matrix metalloproteinases expression

Imbalanced metabolism of Nucleus pulposus (NP) extracellular matrix (ECM) is closely correlated to Intervertebral Disc Degenerative Disease. LIM mineralization protein‐1 (LMP‐1) has been proven to induce sulfated glycosaminoglycan (sGAG) production in NP and have an anti‐inflammatory effect in pre‐osteoclast. However, whether it has any effect on the NP ECM production and degradation under inflammatory stimulation has not been studied. In the current study, a TNF‐α induced cell model was established in vitro. Lentivirus encoding LMP‐1 (LV‐LMP‐1) and short heparin LMP‐1 (LV‐shLMP‐1) were constructed to overexpress and knockdown LMP‐1 expression in NP cells. LMP‐1 mRNA level was regulated in a dose‐dependent manner after transfection. LV‐LMP‐1 increased whereas LV‐shLMP‐1 decreased collagen II, aggrecan, versican expression, and sGAG production. LV‐LMP‐1 abolished while LV‐shLMP‐1 aggravated TNF‐α mediated down‐regulation of the above matrix genes via ERK1/2 activation. Moreover, LV‐LMP‐1 abrogated TNF‐α induced MMP‐3 and MMP‐13 expression via inhibiting p65 translocation and MMP‐3 and MMP‐13 promoter activity. These results indicated that LMP‐1 had an ECM production maintenance effect under inflammatory stimulation. This effect was via up‐regulation of matrix genes expression at least partially through ERK1/2 activation, and down‐regulation of MMPs expression through NF‐κB inhibition.

The effects of punctured nucleus pulposus on lumbar radicular pain in rats: a behavioral and immunohistochemical study.

OBJECT Application of the nucleus pulposus (NP) to the nerve root has been developed as a model of lumbar radicular pain. The relationship between disc degeneration and the induction of radicular pain, however, has not yet been fully explored. The authors of this study investigated pain-related behaviors and expression of tumor necrosis factor-alpha (TNF-alpha) in the dorsal root ganglion (DRG) to evaluate the effects of punctured NP on lumbar radiculopathy. METHODS An anular needle puncture model of intervertebral disc degeneration in a rat tail was established. Normal and previously punctured NP tissues were obtained and placed on the L-5 nerve root following a hemilaminectomy. Behavioral tests including assessment of motor function, mechanical threshold, and thermal withdrawal latency were performed before and after surgery. The TNF-alpha immunoreactivity in L-5 DRG specimens was examined through immunohistochemical study. RESULTS The punctured discs showed significant degeneration 2 weeks after intervention. Application of both normal and punctured NP induced mechanical hyperalgesia in the ipsilateral paw for 10 days after surgery, but hyperalgesia was more severe in the punctured NP group. No statistically significant within-group changes in thermal withdrawal latency over time were found. A significant increase in the expression of TNF-alpha-positive neurons in DRG specimens was observed in both NP graft groups. CONCLUSIONS Needle puncture led to degenerative changes in the rat tail disc, and the degenerated NP enhanced mechanical hyperalgesia induced by application of the NP to the lumbar nerve root. This model of disc degeneration and lumbar radicular pain is appropriate for evaluating the efficacy of biological treatments for degenerative disc diseases.

Both Expression of Cytokines and Posterior Annulus Fibrosus Rupture Are Essential for Pain Behavior Changes Induced by Degenerative Intervertebral Disc: An Experimental Study in Rats

The aim of this study was to analyze the relationship between intervertebral disc degeneration and low back pain (LBP). Rat L4/5 disc degeneration model was established by annular puncture using a 0.4 mm needle anteriorly or posteriorly. In both anterior and posterior puncture models, magnetic resonance imaging (MRI) and histological analyses revealed marked disc degeneration 2 weeks after puncture. Cytokine expression was up‐regulated in different level in nucleus pulposus (NP) from 3 days after puncture. Pain behavioral tests indicated that the anterior disc puncture did not induce pain behavior changes, whereas the posterior disc puncture resulted in mechanical allodynia from 1 day to 21 days after injury. Besides, cytokine expression was significantly increased in dorsal root ganglion (DRG) at 1 and 2 weeks after posterior puncture, but not after the anterior puncture. These findings indicate the NP of the degenerative disc expresses different levels of inflammatory cytokines, and posterior disc puncture produced mechanical allodynia. The expression phase of cytokines in the NP was accordance with mechanical hyperalgesia in the posterior disc puncture model. Both expression of cytokines and posterior annulus fibrosus (AF) rupture in degenerative intervertebral disc are essential for pain behavior changes.

LIM Mineralization Protein-1 Enhances Bone Morphogenetic Protein-2–Mediated Osteogenesis Through Activation of ERK1/2 MAPK Pathway and Upregulation of Runx2 Transactivity

LIM mineralization protein‐1 (LMP‐1) is an intracellular regulator of bone formation. Upregulation of bone morphogenetic proteins (BMPs) and stabilization of BMP/Smad signaling have been proven to be the key mechanisms through which LMP‐1 enhances osteogenesis. However, how LMP‐1 regulates BMPs expression and related bone formation remains unclear. In this study, a LMP‐1–induced osteogenesis cell model was used to study the molecular action of LMP‐1 on BMP‐2 expression and bone formation. The results show that overexpression of LMP‐1 significantly increases, whereas downregulation of endogenous LMP‐1 decreases BMP‐2 expression and bone formation. Antagonism of BMP‐2 with noggin or short hairpin BMP‐2 significantly attenuates the osteoinductive effect of LMP‐1, suggesting that the osteoinductive effect of LMP‐1 is mediated by BMP‐2. LMP‐1 regulation of BMP‐2 is found to occur at the transcription level using a luciferase reporter assay with a reporter construct containing a BMP‐2 promoter. A promoter deletion assay reveals that –1000/–500 bp is the key regulated region by LMP‐1. A Runx2‐binding site is then located at –934/–920 bp and confirmed by luciferase assay using a reporter construct containing repeats of this Runx2‐binding site and the site‐directed mutagenesis analysis. Overexpression of LMP‐1 significantly increases Runx2 expression. Downregulation of Runx2 expression significantly decreases BMP‐2 promoter activity and BMP‐2 expression. A ChIP assay demonstrates that LMP‐1 increases the interaction between Runx2 and BMP‐2 promoter. A luciferase reporter assay using the OSE2 promoter containing a Runx2‐binding site confirms that Runx2 transactivity can be upregulated by LMP‐1. Moreover, inhibiting the activation of different pathways with specific pathway inhibitors reveals that ERK1/2 MAPK activation is essential for LMP‐1–induced upregulation of Runx2 transactivity and subsequent BMP‐2 expression. In conclusion, our novel findings describe a positive regulatory effect of LMP‐1 on BMP‐2 expression and BMP‐2–mediated osteogenesis. This effect occurs through activation of ERK1/2 pathway and subsequent upregulation of Runx2 transactivity.

Hypoxia suppresses serum deprivation‐induced degradation of the nucleus pulposus cell extracellular matrix through the JNK and NF‐κB pathways

Intervertebral disc (IVD) degeneration is associated with the imbalance between anabolism and catabolism of the nucleus pulposus (NP) extracellular matrix (ECM). Serum deprivation (SD) has been reported to exacerbate IVD degeneration; however, the effect of SD on ECM metabolism is not fully understood. Hypoxia plays important roles in maintaining the physiological functions of IVD cells; however, whether hypoxia has any effect on NP ECM production under conditions of SD is still unclear. In the current study, we established an in vitro SD model by exposing NP cells to serum‐free medium. SD decreased the expression of aggrecan and collagen II, as well as the production of sulfated glycosaminoglycan (sGAG) in a time‐dependent manner. However, hypoxia abolished SD‐mediated down‐regulation of aggrecan and collagen II expression via JNK1/2 activation. Moreover, hypoxia abolished SD‐induced MMP‐3 and MMP‐13 expression by inhibiting NF‐κB activation, p65 translocation, and MMP‐3 and MMP‐13 promoter activity. These results indicated that, hypoxia maintained ECM production under conditions of SD. This effect was elicited in part through JNK1/2‐mediated up‐regulation of matrix gene expression and down‐regulation of MMP expression, through the inhibition of NF‐κB.

Inflammation Intensity-Dependent Expression of Osteoinductive Wnt Proteins Is Critical for Ectopic New Bone Formation in Ankylosing Spondylitis

To investigate the molecular mechanism underlying inflammation‐related ectopic new bone formation in ankylosing spondylitis (AS).