Sunao Tanaka

Mesenchymal stem cell isolation and characterization from human spinal ligaments.

Mesenchymal stem cells (MSCs) have a fibroblast-like morphology, multilineage potential, long-term viability and capacity for self-renewal. While several articles describe isolating MSCs from various human tissues, there are no reports of isolating MSCs from human spinal ligaments, and their localization in situ. If MSCs are found in human spinal ligaments, they could be used to investigate hypertrophy or ossification of spinal ligaments. To isolate and characterize MSCs from human spinal ligaments, spinal ligaments were harvested aseptically from eight patients during surgery for lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament. After collagenase digestion, nucleated cells were seeded at an appropriate density to avoid colony-to-colony contact. Cells were cultured in osteogenic, adipogenic or chondrogenic media to evaluate their multilineage differentiation potential. Immunophenotypic analysis of cell surface markers was performed by flow cytometry. Spinal ligaments were processed for immunostaining using MSC-related antibodies. Cells from human spinal ligaments could be extensively expanded with limited senescence. They were able to differentiate into osteogenic, adipogenic or chondrogenic cells. Flow cytometry revealed that their phenotypic characteristics met the minimum criteria of MSCs. Immunohistochemistry revealed the localization of CD90-positive cells in the collagenous matrix of the ligament, and in adjacent small blood vessels. We isolated and expanded MSCs from human spinal ligaments and demonstrated localization of MSCs in spinal ligaments. These cells may play an indispensable role in elucidating the pathogenesis of numerous spinal diseases.

Genetic Differences in the Osteogenic Differentiation Potency According to the Classification of Ossification of the Posterior Longitudinal Ligament of the Cervical Spine

Study Design. We categorized the four types of ossification of the posterior longitudinal ligament (OPLL) of the cervical spine into two groups. We biochemically investigated the genetic differences in the osteogenic differentiation potency between the two groups. Objective. To investigate the genetic differences in the osteogenic differentiation potency according to the OPLL classification. Summary of Background Data. Clinical studies on OPLL have revealed that the risk of progression of the ossification area is greatest for continuous and mixed type OPLL. However, until now, these four types of OPLL have been studied as a single condition. Methods. We categorized the four types of OPLL into the OPLL continuous (continuous or mixed type) and OPLL segmental groups (segmental or circumscribed type). Paraspinal ligaments were aseptically obtained from OPLL patients during surgery. The fibroblast-like cells that migrated from the explants were used for experiments. The cells were placed in a 60-mm culture dishes for total ribonucleic acid preparation and 12 well microplates for alkaline phosphatase (ALP) activity staining. After cultures reached confluence, the cells were cultured in osteogenic medium. The messenger ribonucleic acid expression of bone morphogenetic protein-2 (BMP-2), osterix, tumor necrosis factor-&agr;-stimulated gene-6, and ALP was analyzed by quantitative real time-polymerase chain reaction. Osteogenic differentiation of fibroblast-like cells was determined by histochemically detecting ALP production. Results. After osteogenic induction, BMP-2 expression increased in the OPLL continuous and segmental groups. Osterix expression increased in the OPLL continuous group only. Tumor necrosis factor-&agr;-stimulated gene-6 expression was suppressed in the OPLL continuous and segmental groups. ALP expression as well as ALP activity staining was higher in the OPLL continuous group than in the OPLL segmental group. Conclusion. The study revealed genetic differences in the osteogenic differentiation potency between the OPLL continuous and segmental groups. We propose to distinguish OPLL continuous group from segmental group in biochemical studies on OPLL.

Interobserver and intraobserver reliability of the classification and diagnosis for ossification of the posterior longitudinal ligament of the cervical spine

PurposeOssification of the posterior longitudinal ligament (OPLL) of the cervical spine has been classified into four types by lateral plain radiographs, but the reliability of the classification and of the diagnosis of either cervical OPLL or cervical spondylotic myelopathy (CSM) was unknown. We investigated the interobserver and intraobserver reliability of the classification and diagnosis for OPLL by radiographs and computed tomography (CT) images.MethodsA total of 16 observers classified each patient’s images into five groups; OPLL continuous, segmental, mixed, circumscribed type, or CSM. To evaluate interobserver reliability, the observers first classified only radiograph images, and next both radiographs and CT images. On another day they followed the same procedure to evaluate intraobserver reliability. We also evaluated interobserver and intraobserver reliability of the diagnosis of either cervical OPLL or CSM.ResultsInterobserver reliability of the classification with radiographs only showed moderate agreement, but interobserver reliability with both radiographs and CT images showed substantial agreement. Intraobserver of reliability the classification was also improved by additional CT images. Interobserver reliability of the diagnosis with both radiographs and CT images was almost similar to with radiographs only. Intraobserver reliability of the diagnosis was improved by additional CT images.ConclusionsThis study suggested that the reliability of the classification and diagnosis for cervical OPLL was improved by additional CT images. We propose that diagnostic criteria for OPLL include both radiographs and CT images.

P2Y1 Transient Overexpression Induced Mineralization in Spinal Ligament Cells Derived from Patients with Ossification of the Posterior Longitudinal Ligament of the Cervical Spine

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. We previously reported that P2 purinoceptor Y1 (P2Y1) expression is elevated in the spinal ligament cells of OPLL patients, but the role of P2Y1 in the spinal ligament calcification process is unknown. To verify the hypothesis that P2Y1 expression causes ossification of the spinal ligaments, we forced expression of P2Y1 in spinal ligament cells obtained from OPLL and non-OPLL patients using a cytomegaloviral vector. The expression of mRNA and protein was investigated by quantitative real-time polymerase chain reaction and immunofluorescence staining, respectively. After transfection, bone morphogenetic protein-2 (BMP-2) and Sox9 mRNA expression was significantly increased in spinal ligament cells derived from OPLL patients (4.36- and 6.44-fold, respectively) compared with cells from non-OPLL patients (0.57- and 3.64-fold, respectively) 2xa0days after P2Y1 transient transfection. Furthermore, a statistically significant correlation was observed between BMP-2 and P2Y1 mRNA expression levels in cells obtained from OPLL patients but not from non-OPLL patients. Immunofluorescence analysis showed that BMP-2 and P2Y1 expression was increased in OPLL patients only, while Sox9 expression was increased in OPLL and non-OPLL patients. MRS2279, a selective P2Y1 antagonist, blocked the upregulation of Sox9 and BMP-2 after forced expression of P2Y1. Furthermore, 4xa0days after transient transfection of P2Y1, mineralization was observed only in spinal ligament cells from OPLL patients. These results suggest that P2Y1 expression plays an important role in ectopic bone formation in the spinal ligaments of OPLL patients.

Indications for prophylactic lumbar decompression at the L3/4 level in patients with L4/5 responsible lumbar spinal canal stenosis

Introduction Lumbar spinal canal stenosis (LSS) is a very common disease. When the responsible level is considered to be L4/5 despite the appearance of double-level (L3/4 and L4/5) stenosis on magnetic resonance imaging (MRI), it is difficult for spinal surgeons to decide whether prophylactic decompression should be performed at the L3/4 level. The purpose of this study was to investigate the relationship between the dural sac cross-sectional area (DCSA) at the L3/4 level and clinical symptoms in patients with double-level stenosis. Methods Thirty-five patients with double-level stenosis were registered in this study. All patients underwent decompression surgery at the L4/5 responsible level. The severity of patients symptoms was evaluated by the Japanese Orthopaedic Association (JOA) score and its rate of recovery. A measurement program on MRI was used to determine the DCSA. Results The clinical course of LSS according to the JOA score recovery rate at the final follow-up revealed that the good group (≥50%) included 27 patients, and the poor group (<50%) included 8 patients. In the good group, the mean DCSA at the L3/4 level was 72.3 ± 32.1 mm2 preoperatively and 71.3 ± 29.0 mm2 at the final follow-up. In contrast, in the poor group, the mean DCSA at the L3/4 level was 49.1 ± 23.8 mm2 preoperatively and 40.6 ± 14.1 mm2 at the final follow-up. Significant differences were observed in the preoperative and final follow-up DCSAs at the L3/4 level between two groups. Conclusions Considering the present results, prophylactic decompression surgery at the L3/4 level should be performed for patients with double-level stenosis and DCSA <50 mm2 at the L3/4 level.