Chang-Qing Zhao

The cell biology of intervertebral disc aging and degeneration.

Intervertebral disc degeneration, which mimics disc aging but occurs at an accelerated rate, is considered to be related to neck or low back pain and disc herniation. Degenerated discs show breakdown of the extracellular matrix and thus fail to bear the daily loadings exerted on the spine. Rather than a passive process of wear and tear, disc degeneration is an aberrant, cell-mediated response to progressive structural failure due to aging and other environmental factors such as abnormal mechanical stress. With aging and degeneration, disc cells undergo substantially biologic changes, including alternation of cell type in the nucleus pulposus, increased cell density but decreased number of viable cells as a result of increased cell death and increased cell proliferation, increased cell senescence, and altered cell phenotype which is characterized by compromised capability of synthesizing correct matrix components and by enhanced catabolic metabolism. These changes are involved in the process of disc degeneration through the complicated interactions among them. To retard or reverse disc degeneration, the abnormal conditions of the decreased viable cell population and the altered cell phenotype should be corrected. As potential therapies for disc degeneration, intradiscal protein injection, gene transfer and cell implantation are being understudied in vivo. Suppression of excessive apoptosis and accelerated senescence of disc cells may be other choices for treating disc degeneration. When performing a biologic therapy in order to repair or regenerate the degenerated disc, nutrient and biomechanical factors should also be incorporated, because they are the major causes of the biologic changes experienced by disc cells. Moreover, a very early intervention is indicated by the finding that the onset of human disc degeneration occurs as early as by adolescence.

Modic changes: a systematic review of the literature

Modic changes (MC) are a common phenomenon on magnetic resonance imaging (MRI) in spinal degenerative diseases and strongly linked with low back pain (LBP). Histology, radiology, potential mechanisms, natural history and clinical studies of MC has formed the foundation on which our understanding of spinal degenerative diseases is built. The objective of this study was to provide a review of recent important advances in the study of MC and their clinical significance. This review article summarizes these studies, by delineating the possible mechanisms, and raising doubts and new questions. The related aspects such as discography and differential diagnosis with spinal infection and tumor on MRI are also discussed. Although most of researchers believe that MC are common findings in patients with spinal degenerative diseases and have an association with discogenic LBP, different results between studies may be produced from the differences in study design, inclusion criteria, and sample size. How the present knowledge of MC affects the management of spinal degenerative diseases remains unclear. Further studies of MC will explore therapeutic possibilities for future treatments of spinal degenerative diseases.

Programmed cell death in intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is largely a process of destruction and failure of the extracellular matrix (ECM), and symptomatic IVD degeneration is thought to be one of the leading causes of morbidity or life quality deterioration in the elderly. To date, however, the mechanism of IVD degeneration is still not fully understood. Cellular loss from cell death in the process of IVD degeneration has long been confirmed and considered to contribute to ECM degradation, but the causes and the manners of IVD cell death remain unclear. Programmed cell death (PCD) is executed by an active cellular process and is extensively involved in many physiological and pathological processes, including embryonic development and human degenerative diseases. Thus, the relationship between PCD and IVD degeneration has become a new research focus of interest in recent years. By reviewing the available literature concentrated on PCD in IVD and discussing the methodology of detecting PCD in IVD cells, its inducing factors, the relationship of cell death to ECM degradation, and the potential therapy for IVD degeneration by modulation of PCD, we conclude that IVD cells undergo PCD via different signal transduction pathways in response to different stimuli, that PCD may play a role in the process of IVD degeneration, and that modulation of PCD might be a potential therapeutic strategy for IVD degeneration.

Both endoplasmic reticulum and mitochondria are involved in disc cell apoptosis and intervertebral disc degeneration in rats

Intervertebral disc cell apoptosis occurs through either death receptor or mitochondrial pathway, but whether disc cell apoptosis is also mediated by the endoplasmic reticulum (ER) pathway remains unclear. The objective of this study was to investigate whether ER and mitochondria are co-involved in disc cell apoptosis and intervertebral disc degeneration (IVDD) in rats. Forty-eight rats were used for in vivo experiments. IVDD was characterized by X-ray and histomorphology examination, disc cell apoptosis was detected by TUNEL staining, and the co-involvement of ER and mitochondria in apoptosis was determined by immunohistochemical staining for GRP78, GADD153, caspase-12, and cytochrome C. Additional eight rats were used for annular cell isolation and culture. After sodium nitroprusside treatment, annular cell apoptosis was observed morphologically and quantified by flow cytometry; the expression of biomarkers of ER stress and mitochondrial dysfunction were analyzed by reverse transcriptase PCR (RT-PCR), fluorescence double labeling, and Western blot; and mitochondrial membrane potential was detected by 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbo cyanine iodide (JC-1) staining. Finally, NS3694 and Z-ATAD-FMK were employed to inhibit the formation of apoptosome complex and the activation of caspase-12, respectively, and apoptotic incidence and caspase-9 activity were assayed. We found that IVDD, induced by unbalanced dynamic and static forces in the rats, was accompanied by increased disc cell apoptosis and enhanced expression of GRP78, GADD153, caspase-12, and cytochrome C. Annular cell apoptosis induced by sodium nitroprusside was confirmed by morphologic observation and flow cytometry. With increased apoptosis, the expression of GRP78, GADD153, and caspase-12 upregulated, mitochondrial membrane potential decreased, and accumulation of cytochrome C in the cytosol manifested. Furthermore, NS3694 and Z-ATAD-FMK dramatically suppress annular cell apoptosis and caspase-9 activity. In conclusion, disc cell apoptosis mediated simultaneously by ER and mitochondria plays a potent role in IVDD.

Substrate stiffness regulates apoptosis and the mRNA expression of extracellular matrix regulatory genes in the rat annular cells.

Cells are subjected to static tension of different magnitudes when cultured on substrates with different stiffnesses. It has long been recognized that mechanical stress is an important modulator of the intervertebral disc degeneration. Here we studied the influence of substrate stiffness on cell morphology, apoptosis and extracellular matrix (ECM) metabolism of the rat annulus fibrosus (AF) cells which are known to be mechanosensitive cells. Polyacrylamide gel substrates with three different stiffnesses were prepared by varying the concentration of acrylamide and bisacrylamide, and the elastic modulus of the different gel substrates were measured with atomic force microscopy (AFM). First-passage rat annular cells were cultured on soft, intermediate, rigid substrates or plastics for 24 or 48 h. The percentages of apoptotic cells were detected by flow cytometry and caspase-3 activity, and morphologic changes were visualized by Hoechst 33258 staining and F-actin staining. In addition, the expression of ECM genes (Col1α1, Col2α1, aggrecan, MMP-3, MMP-13 and ADAMTS-5) were analyzed by RT-PCR. The three different substrates had elastic moduli varying between 1±0.23 kPa (soft, 5% gel with 0.06% bis), 32±2.89 kPa (intermediate, 10% gel with 0.13% bis) and 63±3.45 kPa (rigid, 10% gel with 0.26% bis) with a thickness about 60-70 μm. Most of the rat AF cells appeared small and rounded, and lost most of their stress fibers when cultured on soft substrate. There was a significant increase in the percentage of apoptotic cells in the rat AF cells cultured on soft and intermediate substrates relative to those on plastic surface, with a parallel decrease in the area of cell spreading and nucleus. The AF cells grown on intermediate or rigid substrate had reduced expression of Col1α1, Col2α1 and aggrecan and enhanced expression of MMP-3, MMP-13, and ADAMTS-5 at 24h or 48 h, respectively, relative to those cultured on plastic surface. Conversely, we observed an up-regulation of Col2α1 and aggrecan and no change in the gene expression of MMP-3, MMP-13, and ADAMTS-5 in AF cells on soft substrates. Rat AF cells are sensitive to substrate stiffness which can regulate the morphology, growth, apoptosis and ECM metabolism of rat AF cells, thus indicating the importance of substrate choice for cell transplantation and regeneration for the treatment of disc degeneration using tissue-engineering technique.

Effects of spinal cord injury and hindlimb immobilization on sublesional and supralesional bones in young growing rats

Both spinal cord injury (SCI) and hindlimb cast immobilization (HCI) cause reduction in maturation-related bone gain in young rats, but the effects of the two interventions on bone pathophysiology may be different. The objective of this study was to compare the effects of SCI and HCI on the sublesional/supralesional bones and bone turnover indicators in young rats. Forty male Sprague-Dawley rats (six-week-old) were randomized into four groups, with ten rats in each group. The groups were classified as follows: base-line control, age-matched intact control, HCI, and SCI groups. Bone tissues, blood, and urine samples were studied at 4 weeks after treatments. The tibial dry weights and ash weights in SCI were remarkably reduced by 7.5% (dry weights) and 8.2% (ash weights) compared with HCI. SCI rats showed lower areal bone mineral density in the proximal tibiae compared with HCI rats (- 14%). Cortical thickness and cortical area of the tibial midshaft in SCI were lower than HCI (- 23%, - 33% respectively). The bone surface/bone volume, trabecular separation, trabecular number, connectivity of the trabecular network, and structure model index of the proximal tibiae were remarkably different between SCI and HCI groups. In SCI tibiae, the mineralizing surface, mineral apposition rate, and surface-based bone formation rate were significantly higher than HCI groups (12%, 47%, and 29% respectively). In the compression test, the ultimate load, the energy of ultimate load, and Youngs modulus of the proximal tibiae in SCI rats were significantly lower than HCI rats. The serum levels of osteocalcin and the urinary levels of deoxypyridinoline in SCI were higher than those in HCI. There were no significant changes in supralesional bones between SCI and HCI rats. SCI results in a rapid bone loss with more deterioration of trabecular microstructure and cortical bone geometric structure in sublesional bones. High bone turnover rate and low biomechanics strength were found in tibiae in SCI rats. This might be the result of the imbalance of bone resorption and bone formation induced by the impaired neuronal function.

ADAMTS-5 and Intervertebral Disc Degeneration: The Results of Tissue Immunohistochemistry and In Vitro Cell Culture

Matrix metalloproteinases (MMPs) are known to be involved in IVD degeneration by hydrolyzing the extracellular matrix (ECM), especially the collagens. The degradation of proteoglycans, which is another main ECM component in the IVD, however, has not been extensively investigated. This study aimed to determine the expression of ADAMTS‐5 in human herniated intervertebral disc (IVD) tissues and to investigate whether interleukin‐1β (IL‐1β)‐induced expression of ADAMTS‐5 is mediated by nitric oxide (NO). Forty‐five herniated IVDs were harvested and immunostained to determine the distribution and type of ADAMTS‐5 expressing cells. Rat NP cells maintained in alginate beads were treated with IL‐1β, accumulation of NO was detected by Griess reaction, the expression of ADAMTS‐5 and inducible nitric oxide synthase (iNOS) was analyzed by reverse transcriptase‐polymerase chain reaction (RT‐PCR), the content of proteoglycans in alginate beads was visualized by alcian blue staining, and the effect of aminoguanidine on the changes in alginate beads induced by IL‐1β treatment were also examined. Immunohistochemical results from 45 herniated discs showed that ADAMTS‐5‐positive cells are commonly seen in cell clusters, that the percentage of ADAMTS‐5‐positive cells was higher in uncontained herniated discs than in contained ones, and that the percentage of ADAMTS‐5‐positive cells correlated with the age of the patients. IL‐1β treatment resulted in increased accumulation of NO, increased expression of ADAMTS‐5 and iNOS, whereas the accumulation of proteoglycan in alginate beads decreased. Aminoguanidine significantly reversed the changes in alginate beads induced by IL‐1β treatment. We thus suggested that ADAMTS‐5 is probably involved in the process of IVD degeneration, and that IL‐1β‐induced expression of ADAMTS‐5 is mediated by NO.

No advantages of Gamma nail over sliding hip screw in the management of peritrochanteric hip fractures: A meta-analysis of randomized controlled trials

Purpose. There is a lack of consensus regarding the best option for the operative treatment of peritrochanteric fractures. A meta-analysis was performed to assess the surgical outcomes of peritrochanteric fractures. Methods. Only prospective, randomized studies comparing Gamma nail and sliding hip screw (SHS) fixation in adults were included. Eleven studies met the inclusion criteria: the target population consisted of patients with peritrochanteric fractures, while subtrochanteric fractures were not included; the intervention was SHS compared with Gamma nail; the study was a published randomized trial. Each outcome measure tested was assessed for heterogeneity. If significant heterogeneity was present, data from the studies were not combined. If there was no significant heterogeneity, a combined relative risk was calculated using a fixed effects model and a Z-test was performed to test the overall effect. Results. A total of 1344 peritrochanteric fractures entered into the included studies. There were no differences of mortality, cut-out, non-union, re-operation, wound infection, intra-operative fractures of femur, blood loss or surgical time. Conclusions. It seemed that there were no obvious advantages of Gamma nail fixation over SHS fixation in treating peritrochanteric fractures.

Horner Syndrome due to a solitary osteochondroma of C7: a case report and review of the literature.

Study Design. Case report and review of the literature. Objective. To report a 23-year-old woman with osteochondroma of the lower cervical spine who presented with Horner syndrome and to review the relevant literature. Summary of Background Data. Osteochondroma is the most common benign lesion of bone but rarely affects the spine. Methods. Clinical history, routine radiographs, and computed tomography study of the patient were described. A review of the relevant literature was also done. Results. The patient demonstrated a complete disappearance of clinical symptoms on the follow-up examination 60 days after surgery. No patients with Horner syndrome due to a solitary cervical osteochondroma have been previously reported in English-language medical literature. Conclusion. Vertebral involvement of osteochondroma is rare, especially with neurologic compromise. A young patient is presented with a symptomatic solitary osteochondroma of the seventh cervical vertebra who had Horner syndrome. This case report supports surgical intervention of symptomatic osteochondroma of the cervical spine.

Insulin potentiates the proliferation and bone morphogenetic protein-2-induced osteogenic differentiation of rat spinal ligament cells via extracellular signal-regulated kinase and phosphatidylinositol 3-kinase.

Study Design. This study was designed to confirm the correlation of hyperinsulinemia with ossification of posterior longitudinal ligament (OPLL) of the cervical spine in vitro. Objective. To investigate the effects of insulin on the proliferation, collagen synthesis, and osteogenic differentiation of isolated rat spinal ligament cell in the presence or absence of bone morphogenetic protein-2 (BMP-2). Summary of Background Data. Noninsulin-dependent diabetes mellitus is an independent risk factor for the onset of OPLL, but the mechanism is still unknown. We have hypothesized that insulin may exert direct effects on the proliferation and osteogenic differentiation of spinal ligament cells. Methods. Cells isolated from rat spinal ligaments were stimulated by different concentrations of insulin in the presence or absence of BMP-2. The proliferation of the cell was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid (MTT) and direct cell counting and the procollagen type I amino-terminal peptide was measured by enzyme-linked immunosorbent assay. The gene expressions of insulin receptor, alkaline phosphatase, osteocalcin and Runx2 were examined by quantitative reverse transcriptase-polymerase chain reaction. PI3-K/Akt and extracellular signal-regulated kinase (ERK) were analyzed by western blotting. Results. Insulin positively regulated the expression of its receptor in the cells and stimulated the proliferation of the cells in a time- and dose-dependent manner. Insulin alone did not affect the synthesis of procollagen type I amino-terminal peptide synthesis or osteogenesis differentiation in the cells. However, high concentration of insulin (1000 nmol/L) significantly promoted BMP-2-induced alkaline phosphatase expression and activation, which was associated with the up-regulation of PI3-K/Akt and down-regulation of ERK in the cells. Conclusion. Hyperinsulinemia may contribute to the onset and progression of OPLL in subjects with noninsu-lin-dependent diabetes mellitus by stimulating the proliferation and BMP-2-induced osteogenic differentiation of ligament cells via the activation of insulin receptor and PI3-K/Akt pathway and the suppression of ERK.