Toru Asari

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.

Immunohistochemical localization of mesenchymal stem cells in ossified human spinal ligaments.

Mesenchymal stem cells (MSCs) have been isolated from various tissues and used for elucidating the pathogenesis of numerous diseases. In our previous in vitro study, we showed the existence of MSCs in human spinal ligaments and hypothesized that these MSCs contributed to the pathogenesis of ossification of spinal ligaments. The purpose of this study was to use immunohistochemical techniques to analyze the localization of MSCs in ossified human spinal ligaments in situ. Ossified (OLF) or non-ossified ligamentum flavum (non-OLF) samples from the thoracic vertebra were obtained from patients who had undergone posterior spinal surgery. Serial sections were prepared from paraffin-embedded samples, and double immunofluorescence staining was performed using antibodies against markers for MSCs (CD73, CD90 and CD105), endothelial cells (CD31), pericytes (α-smooth muscle actin), and chondrocytes (S100). Immunolocalization of MSCs was observed in the perivascular area and collagenous matrix in spinal ligaments. Markers for MSCs and pericytes were co-expressed in the perivascular area. Compared with non-OLF, OLF had a large amount of neovascularization in the fragmented ligament matrix, and a high accumulation of MSCs around blood vessels. The prevalence of MSCs in OLF within collagenous matrix was significantly higher than that in non-OLF. Chondrocytes near the ossification front in OLF also presented expression of MSC markers. MSCs may contribute to the ectopic ossification process of OLF through endochondral ossification.

Osteogenic lineage commitment of mesenchymal stem cells from patients with ossification of the posterior longitudinal ligament

Ectopic bone formation is thought to be responsible for ossification of the posterior longitudinal ligament of the spine (OPLL). Mesenchymal stem cells (MSCs) were isolated from spinal ligaments and shown to play a key role in the process of ectopic ossification. The purpose of this study was to explore the capacity of these MSCs to undergo lineage commitment and to assess the gene expression changes between these committed and uncommitted MSCs between OPLL and non-OPLL patients. Spinal ligament-derived cells were obtained from OPLL patients or patients with cervical spondylotic myelopathy (non-ossified) for comparison (n=8 in each group). MSCs from the two patient cohorts were evaluated for changes in colony forming ability; osteogenic, adipogenic and chondrogenic differentiation potential; and changes in gene expression following induction with lineage-specific conditions. We show that the osteogenic differentiation potential was significantly higher in MSCs from OPLL patients than in those from non-OPLL patients. In addition, alkaline phosphatase activity and several osteogenic-related genes expressions (bone morphogenetic protein 2, runt-related transcription factor 2 and alkaline phosphatase) were significantly higher in the OPLL group than in the non-OPLL group. However, single cell cloning efficiency, adipogenic and chondrogenic differentiation, and the expression of adipogenic and chondrogenic-related genes were equivalent between MSCs harvested from OPLL and non-OPLL patient samples. These findings suggest an increase in the osteogenic differentiation potential of MSCs from OPLL patients and that this propensity toward the osteogenic lineage may be a causal factor in the ossification in these ligaments.

High Osteogenic Potential of Adipose- and Muscle-derived Mesenchymal Stem Cells in Spinal-ossification Model Mice

Study Design. Basic experiments in a mouse model of ossification of the posterior longitudinal ligament (OPLL). Objective. To assess the osteogenic potential of mesenchymal stem cells (MSCs) obtained from muscle and adipose tissue in Tiptoe-walking (ttw) mice, in which cervical OPLL compresses the spinal cord and causes motor and sensory dysfunction. Summary of Background Data. In humans, MSCs have been implicated in the pathogenesis of cervical OPLL. Cervical OPLL in ttw mice causes chronic compression of the spinal cord. Few studies have compared the MSC osteogenic potential with behavioral changes in an OPLL animal model. Methods. We compared the osteogenic potential and behavioral characteristics of MSCs from ttw mice (4 to 20 weeks old) with those from control wild-type mice (without hyperostosis). Ligament ossification was monitored by micro-computed tomography and pathology; tissues were double stained with fluorescent antibodies against markers for MSCs (CD45 and CD105), at 8 weeks. The Basso Mouse Scale was used to assess motor function, and heat and mechanical tests to assess sensory function. The osteogenic potential of adipose and muscle MSCs was assessed by Alizarin Red S absorbance, staining for osteogenic mineralization, and real-time quantitative polymerase chain reaction for osteogenesis-related genes. Results. Spinal-ligament ossification began in ttw mice at 8 weeks of age, and the ossified area increased with age. Immunofluorescence staining identified MSCs in the ossification area. The ttw mice became hyposensitive at 8 weeks of age, and Basso Mouse Scale scores showed motor-function deficits starting at 12 weeks of age. Alizarin Red S staining for mineralization showed a higher osteogenic potential in the adipose- and muscle-derived MSCs from ttw mice than from wild-type mice at 4, 8, and 20 weeks of age. Real-time quantitative polymerase chain reaction showed that ttw MSCs strongly expressed osteogenesis-related genes. Conclusion. MSCs derived from muscle and adipose tissue in ttw mice had a high osteogenic potential. Level of Evidence: N/A

D-dimer monitoring combined with ultrasonography improves screening for asymptomatic venous thromboembolism in acute spinal cord injury

Objective: We compared screening methods for asymptomatic venous thromboembolism (VTE) in patients with acute spine and spinal cord injuries (SCI). Patients were screened by D-dimer monitoring alone (DS group) or by D-dimer monitoring combined with ultrasonography (DUS group). Design: Prospective cohort study. Setting: One department of a university hospital in Japan. Participants: 114 patients treated for acute SCI between 2011 and 2017. Interventions: N/A. Outcome Measures: D-dimers were measured upon admission and 1, 3, 5, 7, and 14 days thereafter. DUS-group patients also underwent an ultrasound 7 days after admission. If ultrasonography indicated deep venous thrombosis (DVT), or if D-dimer levels increased to ≥ 10 µg/mL, the patient was assessed for VTE, including DVT or pulmonary embolism (PE), by contrast venography. We analyzed the incidence of VTE detected in the DS and DUS groups. Results: In the DS group, D-dimers were elevated (≥ 10 µg/mL) in 15 of 70 patients (21.4%), and 9 of the 15 had asymptomatic VTE (12.9%, DVT 11.4%, PE 5.7%). In the DUS group, one patient developed VTE on day 4, and D-dimers were elevated in 13 of 43 patients (30.2%), ultrasonography indicated DVT in 12 patients (27.9%), and asymptomatic VTE was diagnosed in 12 patients (27.9%, DVT 27.9%, PE 4.7%). The DUS group had a higher incidence of DVT (P = 0.002) and VTE (P = 0.042) than the DS group. Conclusions: Combined D-dimer and ultrasound screening in patients with acute SCI improved the detection of VTE, including PE, compared with D-dimer screening alone.