Shoji Seki

A functional SNP in CILP , encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease

Lumbar disc disease (LDD) is caused by degeneration of intervertebral discs of the lumbar spine. One of the most common musculoskeletal disorders, LDD has strong genetic determinants. Using a case-control association study, we identified a functional SNP (1184T → C, resulting in the amino acid substitution I395T) in CILP, which encodes the cartilage intermediate layer protein, that acts as a modulator of LDD susceptibility. CILP was expressed abundantly in intervertebral discs, and its expression increased as disc degeneration progressed. CILP colocalized with TGF-β1 in clustering chondrocytes and their territorial matrices in intervertebral discs. CILP inhibited TGF-β1–mediated induction of cartilage matrix genes through direct interaction with TGF-β1 and inhibition of TGF-β1 signaling. The susceptibility-associated 1184C allele showed increased binding and inhibition of TGF-β1. Therefore, we conclude that the extracellular matrix protein CILP regulates TGF-β signaling and that this regulation has a crucial role in the etiology and pathogenesis of LDD. Our study also adds to the list of connective tissue diseases that are associated with TGF-β.

Preventive measures for axial symptoms following cervical laminoplasty

We have modified the operative procedure of laminoplasty and changed the postoperative therapy since 1997. In the modified group, several modifications were incorporated. The open door type of cervical en bloc laminoplasty was performed. 1) Bone grafts in the open gap were placed at three levels only. 2) Bone grafting in the hinged side was not performed. 3) The neck collar was worn for only 1 month. 4) The doctors explained the importance of the neck muscle and advised patients to perform early exercise of the posterior neck muscle. The patients who had the original operation and postoperative therapy served as the control (control group). The original operation and postoperative therapy were as follows: Bone grafts from dissected spinous processes were put in the opened laminae and fixed with braided wires or nylon threads. Bone grafts in the open gap were placed to extend from three to five levels. Bone chips were also placed in the hinged side. The orthosis was applied for up to 1 months after surgery, and a neck collar was recommended for 1 additional month. Postoperative neurologic recovery and axial symptoms after cervical laminoplasty were compared between the two groups. There was no statistical difference in postoperative neurologic recovery. However, the incidence of axial symptoms was much lower in the modified group compared with the control group. Radiologic examination showed that postoperative range of motion of the cervical spine in the modified group was significantly well preserved and the number of postoperative fused laminae in the modified group was less than that in the control group. Based on these results, we concluded that the modified method is beneficial for the postoperative status of cervical laminoplasty patients.

Development of a new technique for pedicle screw and Magerl screw insertion using a 3-dimensional image guide.

Study Design. We developed a new technique for cervical pedicle screw and Magerl screw insertion using a 3-dimensional image guide. Objective. In posterior cervical spinal fusion surgery, instrumentation with screws is virtually routine. However, malpositioning of screws is not rare. To avoid complications during cervical pedicle screw and Magerl screw insertion, the authors developed a new technique which is a mold shaped to fit the lamina. Summary of Background Data. Cervical pedicle screw fixation and Magerl screw fixation provide good correction of cervical alignment, rigid fixation, and a high fusion rate. However, malpositioning of screws is not a rare occurrence, and thus the insertion of screws has a potential risk of neurovascular injury. It is necessary to determine a safe insertion procedure for these screws. Methods. Preoperative computed tomographic (CT) scans of 1-mm slice thickness were obtained of the whole surgical area. The CT data were imported into a computer navigation system. We developed a 3-dimensional full-scale model of the patients spine using a rapid prototyping technique from the CT data. Molds of the left and right sides at each vertebra were also constructed. One hole (2.0 mm in diameter and 2.0 cm in length) was made in each mold for the insertion of a screw guide. We performed a simulated surgery using the bone model and the mold before operation in all patients. The mold was firmly attached to the surface of the lamina and the guide wire was inserted using the intraoperative image of lateral vertebra. The proper insertion point, direction, and length of the guide were also confirmed both with the model bone and the image intensifier in the operative field. Then, drilling using a cannulated drill and tapping using a cannulated tapping device were carried out. Eleven consecutive patients who underwent posterior spinal fusion surgery using this technique since 2009 are included. The screw positions in the sagittal and axial planes were evaluated by postoperative CT scan to check for malpositioning. Results. The screw insertion was done in the same manner as the simulated surgery. With the aid of this guide the pedicle screws and Magerl screws could be easily inserted even at the level where the pedicle seemed to be very thin and sclerotic on the CT scan. Postoperative CT scan showed that there were no critical breaches of the screws. Conclusion. This method employing the device using a 3-dimensional image guide seems to be easy and safe to use. The technique may improve the safety of pedicle screw and Magerl screw insertion even in difficult cases with narrow sclerotic pedicles.

Nutlin-3 enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through up-regulation of death receptor 5 (DR5) in human sarcoma HOS cells and human colon cancer HCT116 cells.

MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit the p53-MDM2 interaction and activate p53 signaling. The expressions of DR4 and DR5, Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, are regulated by p53. In this study, the combined effects of nutlin-3 and TRAIL on apoptosis were investigated in HOS and HCT116 cells, which express wild-type p53. Nutlin-3 and TRAIL synergistically enhanced apoptosis owing to their intrinsic and extrinsic pathway signals, respectively. The increase in the Bid expression level and the decrease in the expression levels of anti-apoptotic proteins, c-FLIP and XIAP, were involved in this apoptosis enhancement. Furthermore, nutlin-3 activated the DR5 promoter and increased the expression levels of DR5 at mRNA and protein levels. These results indicate that the combination, treated with nutlin-3 and TRAIL, is useful for apoptosis induction in malignant cells expressing wild-type p53.

Isolation and characterization of human amniotic mesenchymal stem cells and their chondrogenic differentiation.

Background Freshly isolated human amniotic mesenchymal (fHAM) cells contain somatic stem cells possessing proliferative ability and pluripotency, including a chondrogenic lineage. However, little is known about the biology of amnion-derived mesenchymal stem cells (MSCs) because fHAM cells can barely survive to expand under culture conditions in vitro for a long time. Methods In this study, we separated fHAM cells and seeded them to isolate MSCs and analyze its character. In addition, suitable chondrogenic growth factor was determined by pellet culture, and their viability under xenogenic environment was examined by transplantation into rabbit knee joints. Results We succeeded in purifying proliferative subpopulations of fHAM cells, which could continue to proliferate more than 50 cumulative population doubling levels, and designated them as HAM&agr; cells. Flow cytometry analysis revealed that they were positive for MSC markers (CD44, CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD34, CD14, and CD45) and major histocompatibility complex class II antigen (human leukocyte antigen–DR). The expression of various stem-cell markers such as OCT3/4, C-MYC, SOX2, NANOG, CD44, SSEA-3, and SSEA-4 was also proved by immunocytochemical staining. Pellet culture using chondrogenic medium supplemented with transforming growth factor &bgr;3, transforming growth factor &bgr;3 plus bone morphogenetic protein (BMP)-2, or BMP-2 implied that supplementation of BMP-2 alone most effectively induced chondrogenesis in vitro. Xenotransplantation of HAM&agr; cells achieved 8-week survival in vivo. Conclusions These results suggest that HAM&agr; cells correspond to MSCs that are highly proliferative and multipotent. Their chondrogenic potential and low immunogenicity indicate that HAM&agr; cells could be an allotransplantable cell resource for cartilage repair.

Ionizing Radiation Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis through Up-Regulations of Death Receptor 4 (DR4) and Death Receptor 5 (DR5) in Human Osteosarcoma Cells

Despite improvements in chemotherapy and surgery in the treatment of osteosarcoma (OS), satisfactory results are still difficult to achieve. Novel therapeutic modalities need to be developed for osteosarcoma treatment. The combined effects of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and ionizing radiation (IR) on human OS cells were investigated. IR and TRAIL treatment synergistically decreased the cell viability and enhanced apoptosis in OS cell lines. IR pretreatment enhances TRAIL‐induced Bid and caspase‐3 activations. Decreases in the expression levels of the antiapoptotic proteins c‐FLIP and XIAP also associated with apoptosis enhancement. Furthermore, IR pretreatment enhanced DR4 and DR5 expressions at the transcription stage. These results can become the basic lines of evidence for the future treatment of OS using TRAIL with IR.

Ossification of the posterior longitudinal ligament in not only the cervical spine, but also other spinal regions: analysis using multidetector computed tomography of the whole spine.

Study Design. A prospective cohort study. Objective. To evaluate ossification of the posterior longitudinal ligament (OPLL) of the whole spine in patients with cervical OPLL and to analyze which types of cervical OPLL were associated with the other lesions in the thoracic and/or lumbar spine. Summary of Background Data. OPLL is most frequently seen in the cervical spine. The coexisting ossified lesions are sometimes observed in other spinal regions. However, coexisting OPLL in other spinal regions have not yet been precisely evaluated in patients with cervical OPLL. Methods. One hundred seventy-eight patients with a diagnosis of cervical OPLL whose plain radiographs were obtained were included. Computed tomographic images of the whole spine were obtained. The ossification index (OS index) was newly determined according to the sum of the levels of vertebral bodies and intervertebral discs with OPLL. The patients were divided into 2 groups, the group that had OPLL only in the cervical spine (C group) and the group that had OPLL in multilevel spinal regions other than the cervical spine (M group). Results. Ninety-five (53.4%) had OPLL not only in the cervical spine, but also in other spinal regions. The M group had more females than the C group. The incidence of bridge formation in the cervical spine was higher in M group than in C group. More females had a high OS index. A positive correlation was found between the OS index of the cervical spine and the OS index of the thoracic and lumbar spine; however, the r value was small. Conclusion. This study demonstrated that more than half of the patients with cervical OPLL had coexisting OPLL in the thoracic and/or lumbar spine. We strongly recommend computed tomographic analysis of the whole spine for patients with radiographical evidence of OPLL in the cervical spine for the early detection of additional sites of ossification. Level of Evidence: 4

Multi-focal Myxopapillary Ependymoma in the Lumbar and Sacral Regions Requiring Cranio-spinal Radiation Therapy: A Case Report

Ependymomas are uncommon tumors that arise in the brain, spinal cord or cauda equina. Myxopapillary ependymomas is located exclusively in the conus medullaris or cauda equina, or film terminale region. In most myxopapillary ependymomas, the histological examination reveals low mitotic activity that is associated with a low MIB-1 labeling index (LI). The prognosis is generally favorable, when the appropriate treatment, including a total resection, is performed. The authors encountered a 39-year-old man with multifocal type of myxopapillary ependymomas compressing the cauda equina from the L2 to L3 level and L5-S1 level. A subtotal resection of the tumor was carried out. The histological examination revealed extremely high mitotic activity with a MIB-1 LI of 9.1%. Therefore, cranio-spinal radiation was added after surgery. The postoperative course was uneventful over the 3.5 year follow-up period.

Cartilage intermediate layer protein promotes lumbar disc degeneration

Lumbar disc disease (LDD) is one of the most common musculoskeletal disorders, and accompanies intervertebral disc degeneration. CILP encodes cartilage intermediate layer protein, which is highly associated with LDD. Moreover, CILP inhibits transcriptional activation of cartilage matrix genes in nucleus pulposus (NP) cells in vitro by binding to TGF-β1 and inhibiting the phosphorylation of Smads. However, the aetiology and mechanism of pathogenesis of LDD in vivo are unknown. To demonstrate the role of CILP in LDD in vivo, we generated transgenic mice that express CILP specifically in the intervertebral disc tissues and assessed whether CILP exacerbates disc degeneration. Degeneration of the intervertebral discs was assessed using magnetic resonance imaging (MRI) and histology. The level of phosphorylation of Smad2/3 in intervertebral discs was measured to determine whether overexpressed CILP suppressed TGF-beta signalling. Although the macroscopic skeletal phenotype of transgenic mice appeared normal, histological findings revealed significant degeneration of lumbar discs. MRI analysis of the lumbar intervertebral discs indicated a significantly lower signal intensity of the nucleus pulposus where CILP was overexpressed. Intervertebral disc degeneration was also observed. The number of phosphorylation of Smad2/3 immuno-positive cells in the NP significantly was decreased in CILP transgenic mice compared with normal mice. In summary, overexpression of CILP in the NP promotes disc degeneration, indicating that CILP plays a direct role in the pathogenesis of LDD.

Variables affecting postsurgical prognosis of thoracic myelopathy caused by ossification of the ligamentum flavum

BACKGROUND CONTEXT Ossification of the ligamentum flavum (OLF) may result in thoracic myelopathy (TM) because of narrowing of the spinal canal. Because symptoms vary and are subjective, diagnosis of TM caused by OLF is sometimes difficult when based on symptoms and physical examination. Posterior decompression is indicated in patients with TM caused by OLF because it is believed that surgery is the most effective treatment. However, surgical outcomes vary. We are unaware of reports of objective presurgical diagnostic parameters, such as neurologic and radiologic findings, relating to the postsurgical prognosis in patients with TM caused by OLF. PURPOSE To determine which presurgical and surgical variables were most closely related to postsurgical prognosis of TM caused by OLF. STUDY DESIGN Retrospective review of the records of the cohort of patients who had undergone surgery from 1988 through 2008 at the University of Toyama Hospital for TM caused by OLF. PATIENT SAMPLE Forty-one patients who had surgery for TM caused by OLF that was progressive, severe, or both and for which the diagnosis was based on clinical, radiologic, and pathologic evaluations. OUTCOME MEASURES Relationship between the highest follow-up Japanese Orthopaedic Association (JOA) score for neurologic evaluation and of Hirabayashis formula to indicate the extent of normalization after surgery with respect to the following 10 variables: age at surgery; sex; duration of presurgical symptoms; complications of diabetes mellitus; complications of hypertension; presence of presurgical hyperreflexia in either or both of the patellar tendon reflex and the Achilles tendon reflex; presurgical impairment of joint position sense in the big toes; number of levels affected by OLF; concurrent spinal lesions including ossification of the posterior longitudinal ligament; and intramedullary change of the spinal cord seen on magnetic resonance imaging (MRI). METHODS Multiple linear analyses were used to evaluate the variables related to postsurgical recovery. RESULTS Presurgical impairment of joint position sense in the big toe was the most important predictor of the highest postsurgical JOA score and of the highest percentage recovery rate. The number of affected OLF levels also predicted the postsurgical highest JOA score, but not statistically significantly so. Age at surgery, sex, and duration of symptoms presurgically did not affect postsurgical recovery. Complications of diabetes mellitus or of hypertension did not affect percentage recovery rate. The difference between recovery rate in patients with or without concurrent spinal lesions was not significant. Presurgical hyperreflexia was not correlated with recovery. Postsurgical JOA scores and percentage recovery rates of scores in patients whose presurgical MRIs had shown intramedullary signal change were not statistically significantly different from those whose MRIs had not shown signal change. CONCLUSIONS An excellent postoperative prognosis is not always possible in patients with TM caused by OLF. It may be important to check for impairment of joint position sense in the big toe, the number of levels affected by OLF, and presurgical intramedullary signal change on MRI before continuing to surgery.