Zhaoqing Guo

Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP(2) in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP(2) expression in BMSCs.

Cbfa1/osf2 Transduced Bone Marrow Stromal Cells Facilitate Bone Formation In Vitro and In Vivo

It has been well established that core binding factor a-1/osteoblast-specific factor-2 (cbfa1/osf2) is a key regulator of osteoblast differentiation and function, however, it is not known whether it can induce bone formation in vitro and in vivo. To investigate the effect of cbfa1/osf2 on bone formation, we used a recombinant adenoviral vector carrying the mouse cbfa1/osf2 gene to transduce primary cultured bone marrow stromal cells (MSCs) of BALB/c mice. We found that Ad-cbfa1/osf2-transduced MSCs produced cbfa1/osf2 protein and differentiated into osteoblast-like cells. The transduced MSCs had increased alkaline phosphatase activity, increased expression of osteocalcin, osteopontin and bone sialoprotein, and increased matrix mineralization in vitro. To observe the induction of bone formation in vivo, MSCs transduced with Ad-cbfa1/osf2 were transplanted into a 5 mm diameter critical-sized skull defect in BALB/c mice, with type I collagen as scaffolding material. Healing of the defect in treatment and control groups was examined grossly and histologically at four weeks. Skull defects transplanted with Ad-cbfa1/osf2-transduced MSCs had an average of 85% osseous closure at four weeks. Control groups in which the defects were not treated (group 1), treated with collagen only (group 2), or treated with collagen and nontransduced MSCs (group 3) showed little or no osseous healing. These studies indicate that cbfa1/osf2 can induce osteoblast differentiation and bone formation both in vitro and in vivo, suggesting that MSCs transduced with the cbfa1/osf2 gene may be useful in treating bone defects.

The frequency and treatment of dural tears and cerebrospinal fluid leakage in 266 patients with thoracic myelopathy caused by ossification of the ligamentum flavum.

Study Design. Retrospective review. Objective. To perform a single-institution analysis of incidence, treatment, and clinical outcome in patients with thoracic ossification of the ligamentum flavum (OLF) who experienced dural tears and cerebrospinal fluid (CSF) leakage. Summary of Background Data. There is a paucity of clinical reports focusing on dural tears and CSF leakage after thoracic OLF surgery. Because dural adhesion and dural ossification are common features of thoracic OLF, the incidence of CSF leakage in OLF patients is high and represents a significant clinical challenge. Methods. A total of 266 patients with thoracic OLF were admitted to our hospital from 1995 to 2011. Each patients medical records were reviewed to identify cases of dural tears and CSF leakage. Information on therapeutic strategy used to repair the dural tears and complications related to CSF leakage was extracted. Results. The incidence of dural tears and CSF leakage in OLF patients was 32% (85/266). The incidence of dural ossification was 25.2%. The dural tears were repaired with a range of materials, including gelatin sponge, muscle/fascia, artificial dura, silk suture, and fibrin glue. The intraoperative repair procedure did not resolve CSF leakage in 65 cases, and 16 of those cases experienced complications related to the continued CSF leakage, including CSF pseudocyst, wound dehiscence, and meningitis. Fifty-eight patients with CSF leakage were eventually cured by a series of comprehensive treatments, which included prone position, continuous pressure by sandbag, ultrasound-guided puncture, and aspiration. Only 7 patients required reoperation. Conclusion. Dural ossification was the main reason for dural tears. In all, 78 of the 85 patients with CSF leakage or dural tear were successfully cured. The success rate was 91.8%, which indicated that a series of comprehensive treatments was an effective strategy to treat these patients.

Analysis of spinopelvic sagittal alignment in patients with thoracic and thoracolumbar angular kyphosis.

Study Design. Retrospective and radiological analysis of spinopelvic sagittal alignment in Chinese patients with thoracic and thoracolumbar kyphosis. Objective. To determine the impact of thoracic and thoracolumbar kyphosis on pelvic sagittal morphology and the mechanisms of adjusting trunk sagittal balance. Summary of Background Data. Previous studies have reported the normative values of pelvic sagittal parameters and classification of normal patterns of sagittal curvature, but no study has analyzed the impact of thoracic and thoracolumbar kyphosis on pelvic sagittal morphology and the mechanisms of maintaining the sagittal balance. Methods. Whole spine and standing lateral radiographs of 49 Chinese patients with thoracic and thoracolumbar kyphosis were obtained before surgery, immediately after surgery, and in the final follow-up. The pelvic and spinal parameters were measured and the correlations of all parameters were analyzed. A descriptive analysis characterizing these parameters and a multivariate analysis were performed. Results. The patients had a mean age of 30.3 years, whereas the mean age at which the patients who developed kyphosis was 7.1 years. Preoperative pelvic incidence was significantly less than that of normal subjects, and there was no difference in the preoperative, in the immediate postoperative, and in the final follow-up radiographs. The magnitude of kyphosis and the levels involved were independent factors of pelvic incidence. Pelvis anteversion and lumbar hyperlordosis were the mechanisms of adjusting the trunk sagittal balance. Although kyphosis and sagittal imbalance was corrected by surgery, pelvic sagittal morphology remained unchanged. Conclusion. Thoracic and thoracolumbar angular kyphosis occurring during the growth period will lead to abnormal pelvic morphology. The greater the kyphotic angle and lower the kyphotic levels, the greater the impact on the pelvic morphology during skeletal maturation. The mechanisms of adjusting the trunk sagittal balance not only include pelvis anteverting, but also lumbar hyperlordosis. The latter serves as the main mechanism once skeletal maturation has been established. After skeletal maturation, surgery can re-establish the spinal sagittal balance but not the pelvis morphology. Level of Evidence: 3

Tissue transglutaminase expression and activity in human ligamentum flavum cells derived from thoracic ossification of ligamentum flavum.

Study Design. The study was undertaken to compare the expression and activity of tissue transglutaminase (TG2) in human ligamentum flavum cells derived from ossification of the ligamentum flavum (OLF) and non-OLF patients. Objective. To determine whether TG2 is involved in the pathologic process of OLF. Summary of Background Data. OLF is a disease characterized by heterotopic formation of new bone in the flavum ligament. Recently, TG2 is proved to directly promote skeletal matrix mineralization and play an important role in the ossification. TG2 activity is vital to the differentiation of osteoblasts and the formation of mineralization. But whether TG2 is involved in the pathologic process of OLF is unknown. We investigated the relations between TG2 expression and OLF. Methods. OLF and non-OLF cells were cultured and osteocalcin, bone morphogenetic protein-2(BMP-2) and TG2 mRNA expressions were assayed by reverse transcription polymerase chain reaction. Meanwhile, alkaline phosphatase activity and calcified nodules were compared between OLF and non-OLF cells. To detect TG2 expression, Western blot and immunohistochemical analysis were carried out, and TG2 activity was compared between OLF and non-OLF cells. Results. Our experiments demonstrated that OLF cells showed osteoblast-like activity and increased mRNA expression of BMP-2. More interesting, compared with non-OLF cells, OLF cells showed elevated expression levels of TG2 mRNA and protein, as well as enzyme activity. Conclusion. TG2 expression and enzyme activity are upregulated in the OLF cells and TG2 may be involved in the pathologic process of OLF.

MiR‐199b‐5p inhibits osteogenic differentiation in ligamentum flavum cells by targeting JAG1 and modulating the Notch signalling pathway

Ossification of the ligamentum flavum (OLF) is a pathology almost only reported in East Asian countries. The leading cause of OLF is thoracic spinal canal stenosis and myelopathy. In this study, the role of miR‐199b‐5p and jagged 1 (JAG1) in primary ligamentum flavum cell osteogenesis was examined. MiR‐199b‐5p was found to be down‐regulated during osteogenic differentiation in ligamentum flavum cells, while miR‐199b‐5p overexpression inhibited osteogenic differentiation. In addition, JAG1 was found to be up‐regulated during osteogenic differentiation in ligamentum flavum cells, while JAG1 knockdown via RNA interference caused an inhibition of Notch signalling and osteogenic differentiation. Moreover, target prediction analysis and dual luciferase reporter assays supported the notion that JAG1 was a direct target of miR‐199b‐5p, with miR‐199b‐5p found to down‐regulate both JAG1 and Notch. Further, JAG1 knockdown was demonstrated to block the effect of miR‐199b‐5p inhibition. These findings imply that miR‐199b‐5p performs an inhibitory role in osteogenic differentiation in ligamentum flavum cells by potentially targeting JAG1 and influencing the Notch signalling pathway.

Polymicrobial and Monomicrobial Infections after Spinal Surgery: A Retrospective Study to Determine which Infection is more Severe

Study Design A retrospective clinical review. Purpose To investigate the difference in clinical manifestations and severity between polymicrobial and monomicrobial infections after spinal surgery. Overview of Literature Surgical site infections (SSIs) after spinal surgery are a major diagnostic and therapeutic challenge for spinal surgeons. Polymicrobial infections after spinal surgery seem to result in poorer outcomes than monomicrobial infections because of complementary resistance to antibiotics. However, comparison of the clinical manifestations and severity between polymicrobial and monomicrobial infections are limited. Methods Sixty-seven patients with SSIs after spinal surgery were studied: 20 patients with polymicrobial infections and 47 with monomicrobial infections. Pathogenic bacteria identified were counted and classified. Age, sex, and body mass index were compared between the two groups to identify homogeneity. The groups were compared for clinical manifestations by surgical site, postoperative time to infection, infection site, incisional drainage, incisional swelling, incisional pain, neurological signs, temperature, white blood cell count, and the percentage of neutrophils. Finally, the groups were compared for severity by hospital stay, number of rehospitalizations, number of debridements, duration of antibiotics administration, number of antibiotics administered, and implant removal. Results Polymicrobial infections comprised 29.9% of SSIs after spinal surgery, and most polymicrobial infections (70.0%) were caused by two species of bacteria only. There was no difference between the groups in terms of clinical manifestations and severity. In total, 96 bacterial strains were isolated from the spinal wounds: 60 strains were gram-positive and 36 were gram-negative pathogenic bacteria. Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Enterobacter cloacae were cultured in order of the frequency of appearance. Conclusions Most polymicrobial infections were caused by two bacterial species after spinal surgery. There was no difference in clinical manifestations or severity between polymicrobial and monomicrobial infections.