Facilitation of Chemotaxis Activity of Mesenchymal Stem Cells via Stromal Cell-Derived Factor-1 and Its Receptor May Promote Ectopic Ossification of Human Spinal Ligaments.

Mesenchymal stem cells (MSCs) have been used to elucidate the pathogenesis of numerous diseases. Our recent study showed that MSCs may conduce to the ossification of spinal ligaments. Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) regulate MSC migration. Moreover, their expression is elevated in sites of damage and remodeling in pathologic states. We explored the possible role of the SDF-1/CXCR4 axis in the chemotactic behavior of MSCs in the ossification of spinal ligaments. Specimens of thoracic vertebra ossified ligamentum flavum (OLF) and non-OLF plaques were received from patients in whom we had performed spine surgery. Paraffin-embedded tissue sections were prepared for immunohistochemical staining. Cultured MSCs from the ligamentum flavum were prepared for in vitro analyses. We observed SDF-1 and CXCR4 localization immunohistochemically in the perivascular area and collagenous matrix of ligaments and in chondrocytes near the ossification front of OLF. And then, immunohistochemical staining showed a close relationship between MSCs and the SDF-1/CXCR4 axis. In the in vitro analyses, expression of the SDF-1/CXCR4 and the migratory capacity of MSCs in OLF were remarkably higher compared with non-OLF MSCs. Furthermore, the migration of MSCs was upregulated by SDF-1 and downregulated by treatment with AMD3100 (C28H54N88HCl), a specific antagonist for CXCR4. All in vitro test data showed a significant difference in MSCs from OLF compared with non-OLF MSCs. Our results reveal that the SDF-1/CXCR4 axis may contribute to an MSC-mediated increase in the ossification process, indicating that the SDF-1/CXCR4 axis may become a potential target for a novel therapeutic strategy for ossification of spinal ligaments.

K-line (-) in the Neck-Flexed Position in Patients With Ossification of the Posterior Longitudinal Ligament Is a Risk Factor for Poor Clinical Outcome After Cervical Laminoplasty.

STUDY DESIGN: Retrospective study comparing postoperative clinical outcomes after cervical laminoplasty between K-line (-) ossification of the posterior longitudinal ligament (OPLL) and K-line (+) OPLL in the neck-flexed position. OBJECTIVE: To investigate postoperative outcomes using Japanese Orthopedic Association (JOA) scores, and grip-and-release (GR) and foot-tap (FT) test scores after laminoplasty in patients with K-line (-) OPLL in the neck-flexed position. SUMMARY OF BACKGROUND DATA: Cervical laminoplasty has been reported to lead to poor outcomes in K-line (-) OPLL and good outcomes in K-line (+) OPLL. The cervical spine, however, continues moving in the extension and flexion direction after laminoplasty. METHODS: Patients with cervical myelopathy were divided into K-line (+) and (-) in the neck-flexed position. We compared postoperative outcomes after cervical laminoplasty using recovery rate, as assessed by the JOA score and degree of improvement in the six JOA score items, and performance, as assessed by GR and (FT) tests, between patients with K-line (+) OPLL (n = 18) and K-line (-) OPLL (n = 23) in the neck-flexed position. RESULTS: Recovery rate of JOA score (23.8%) of patients in the K-line (-) group was significantly lower (P = 0.028) than that (46.3%) of K-line (+) group in the neck-flexed position. In the K-line (+) group, significant improvements were seen in all JOA-score items except bladder function; however, in the K-line (-) group, improvements were seen only in upper- and the lower-extremity sensory functions. In the K-line (+) group, mean GR and FT tests significantly improved, but in the K-line (-) group, only mean FT test significantly improved. CONCLUSION: The K-line (-) OPLL in the neck-flexed position is a risk factor for poor clinical outcome after cervical laminoplasty. LEVEL OF EVIDENCE: 4.

Decreased DNA methylation in the promoter region of the WNT5A and GDNF genes may promote the osteogenicity of mesenchymal stem cells from patients with ossified spinal ligaments.

Mesenchymal stem cells (MSCs) isolated from spinal ligaments with ectopic ossification have a propensity toward the osteogenic lineage. To explore epigenetic control of the osteogenic features of MSCs, we treated MSCs obtained from the spinal ligaments of ossification of yellow ligament (OYL) patients and non-OYL patients with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AdC). We compared the non-OYL groups (untreated and treated with 5AdC) with the OYL groups (untreated and treated with 5AdC) by genome-wide microarray analysis. Next, we used methylated DNA immunoprecipitation combined with quantitative real-time PCR to assess gene methylation. Ninety-eight genes showed expression significantly increased by 5AdC treatment in MSCs from non-OYL patients but not from OYL patients. In contrast, only two genes, GDNF and WNT5A, showed significantly higher expression in OYL MSCs compared with non-OYL MSCs without 5AdC treatment. Both genes were hypermethylated in non-OYL MSCs but not in OYL MSCs. Small interfering RNA targeted to each gene decreased expression of the target gene and also several osteogenic genes. Both small interfering RNAs also suppressed the activity of alkaline phosphatase, a typical marker of osteogenesis. These results suggest that the osteogenic features of MSCs from OYL patients are promoted by unmethylated WNT5A and GDNF genes.

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.

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.