Exosomal miR-140-5p inhibits osteogenesis by targeting IGF1R and regulating the mTOR pathway in ossification of the posterior longitudinal ligament.

BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) is a disabling disease whose pathogenesis is still unclear, and there are no effective cures or prevention methods. Exosomal miRNA plays an important role in the osteogenesis of ectopic bone. Therefore, we focused on the downregulation of miR-140-5p in OPLL cell-derived exosomes to explore the mechanism by which exosomal miR-140-5p inhibits osteogenesis in OPLL. RESULTS: Exosomes were isolated by differential centrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis, and exosomal markers. Exosomal RNA was extracted to perform miRNA sequencing and disclose the differentially expressed miRNAs, among which miR-140-5p was significantly downregulated. Confocal microscopy was used to trace the exosomal miR-140-5p delivered from OPLL cells to human mesenchymal stem cells (hMSCs). In vitro, we verified that exosomal miR-140-5p inhibited the osteoblast differentiation of hMSCs by targeting IGF1R and suppressing the phosphorylation of the IRS1/PI3K/Akt/mTOR pathway. In vivo, we verified that exosomal miR-140-5p inhibited ectopic bone formation in mice as assessed by micro-CT and immunohistochemistry. CONCLUSIONS: We found that exosomal miR-140-5p could inhibit the osteogenic differentiation of hMSCs by targeting IGF1R and regulating the mTOR pathway, prompting a further potential means of drug treatment and a possible target for molecular therapy of OPLL.

The Effect of the NFκB-USP9X-Cx43 Axis on the Dynamic Balance of Bone Formation/Degradation during Ossification of the Posterior Longitudinal Ligament of the Cervical Spine.

Connexin 43- (Cx43-) mediated nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling has been found involved in the ossification of the posterior longitudinal ligament (OPLL). However, the underlying mechanism how OPLL is regulated has not been elucidated. In the present study, primary ligament fibroblast were isolated; immunoprecipitation (IP) and liquid chromatography-mass spectrometry (LC-MS) assays were applied to identify potential binding proteins of Cx43. Protein interaction was then confirmed by co-IP assay. Alkaline phosphatase (ALP) activity and alizarin red staining were used to evaluate ossification. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were employed to assess the binding between NF-κB p65 and target gene. Lipoxygenase inhibitor (5,8,11-eicosatriynoic acid, EPA) was applied to induce endoplasmic reticulum (ER) stress, and 4-phenylbutyrate (4-PBA) was used as an ER-stress inhibitor. Expression of USP9X, Cx43, and nuclei p65 in ligaments from patients and controls was detected by Western blotting. The results showed that ubiquitin-specific protease 9 X-chromosome (USP9X), a deubiquitylating enzyme, was a candidate of Cx43 binding proteins, and USP9X inhibited Cx43 ubiquitination. In vitro experiments showed that USP9X promoted ossification of primary ligament fibroblasts and nuclear translocation of NF-κB p65 by regulating Cx43 expression. Moreover, NF-κB can bind to the USP9X promoter to promote its transcription. When ER stress was inhibited by 4-PBA, USP9X levels, NF-κB nuclei translocation, and ALP activity were decreased. Reverse results were obtained when ER stress was induced by EPA. PDTC, an NF-κB inhibitor, could abolish the effects of EPA. Furthermore, USP9X, Cx43, and nuclei p65 were significantly upregulated in ligaments from OPLL patients than non-OPLL controls. USP9X was positively correlated with CX43 and nuclei p65 in OPLL samples. Overall, the findings suggest that the ER stress-NFκB-USP9X-Cx43 signaling pathway plays an important role in OPLL progression.

Hsa-circ-0007292 promotes the osteogenic differentiation of posterior longitudinal ligament cells via regulating SATB2 by sponging miR-508-3p.

Ossification of the posterior longitudinal ligament (OPLL) is a disorder with multiple pathogenic mechanisms and leads to different degrees of neurological symptoms. Recent studies have revealed that non-coding RNA (ncRNA), including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), could influence the development of OPLL. Nevertheless, the molecular mechanisms linking circular RNAs (circRNAs) and the progression of OPLL is still unknown. The current research explored the expression profiles of OPLL-related circRNAs by microarray analysis, and applied qRT-PCR to validate the results. Subsequently, we confirmed the upregulation of hsa_circ_0007292 in OPLL cells by qRT-PCR and validated the circular characteristic of hsa_circ_0007292 by Sanger sequencing. Fluorescence in situ hybridization (FISH) unveiled that hsa_circ_0007292 was predominantly located in the cytoplasm. Functionally, gain-of-function and loss-of-function experiments showed that hsa_circ_0007292 promoted the osteogenic differentiation of OPLL cells. Mechanistically, the interaction of hsa_circ_0007292 and miR-508-3p was predicted and validated by bioinformatics analysis, dual-luciferase reporter assays, and Ago2 RNA immunoprecipitation (RIP). Similarly, we validated the correlation between miR-508-3p and SATB2. Furthermore, rescue experiments were performed to prove that hsa_circ_0007292 acted as a sponge for miR-508-3p, and SATB2 was revealed to be the target gene of miR-508-3p. In conclusion, our research shows that hsa_circ_0007292 regulates OPLL progression by the miR-508-3p/SATB2 pathway. Our results indicate that hsa_circ_0007292 can be used as a promising therapeutic target for patients with OPLL.

The roles of autophagy in osteogenic differentiation in rat ligamentum fibroblasts: Evidence and possible implications.

Autophagy, a macromolecular degradation process, plays a pivotal role in cell differentiation and survival. This study was designed to investigate the role of autophagy in the osteogenic differentiation in ligamentum fibroblasts. Rat ligamentum fibroblasts were isolated from the posterior longitudinal ligament and cultured in osteogenic induction medium. Ultrastructural analysis, immunofluorescence assay, western blot, flow cytometry, and lysosomal activity assessment were performed to determine the presence and activity of autophagy in the cells. The mineralization deposit and osteogenic gene expressions were evaluated to classify the association between autophagy activity and the bone formation ability of the spinal ligament cells. The influence of leptin and endothelin-1 on the autophagy activity was also evaluated. Our study demonstrated that autophagy was present and increased in the ligament cells under osteogenic induction. Inhibition of autophagy with either pharmacologic inhibitors (Bafilomycin A and 3-methyladenine) or Belcin1 (BECN1) knocking down weakened the mineralization capacity, decreased the gene expressions of COL1A1, osteocalcin (Ocn), and runt-related transcription factor 2 (Runx2) in the ligamentum fibroblasts and increased cell apoptosis. The Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-BECN1 autophagic pathway was activated in the osteogenic differentiating ligamentum fibroblasts. Leptin significantly increased the autophagy activity in the ligament cells under osteogenic induction. These discoveries might improve our understanding for the mechanism of ossification of the posterior longitudinal ligament (OPLL) and provide new approaches on the prevention and treatment of this not uncommon disease.

The COL6A1 rs201153092 single nucleotide polymorphism, associates with thoracic ossification of the posterior longitudinal ligament.

Thoracic ossification of the posterior longitudinal ligament (T­OPLL) is one of the most common factors that causes thoracic spinal stenosis, resulting in intractable myelopathy and radiculopathy. Our previous study reported that the rs201153092 polymorphism present in the collagen 6A1 (COL6A1) gene was a potentially pathogenic locus for the development of T­OPLL. The present study aimed to determine whether the rs201153092 mutation causes abnormal expression of COL6A1 in Han Chinese patients with T­OPLL, and to examine the effects of this mutation on osteogenesis by establishing a model of osteogenic differentiation. COL6A1 gene mutant and wild­type mouse 3T3­E1 embryonic osteoblast models were constructed to induce the differentiation of these cells into osteoblasts. The potential of the mutation site to induce abnormal expression of the COL6A1 gene and osteogenic markers was assessed via reverse transcription­quantitative PCR and western blot analyses. The results demonstrated that the rs201153092A mutation site resulted in significantly increased COL6A1 gene expression levels in the OPLL tissues obtained following clinical surgery. This mutation was shown to play an important role in the development of T­OPLL by regulating the overexpression of the COL6A1 gene and significantly increasing the expression levels of osteogenic markers. The findings of the present study suggested that the rs201153092A mutant variant could increase the expression levels of COL6A1 and consequently play a role in the pathogenesis of T­OPLL.

Roles and mechanisms of leptin in osteogenic stimulation in cervical ossification of the posterior longitudinal ligament.

BACKGROUND: Hyperleptinemia is a common feature of obese people, and leptin, an adipocyte-derived cytokine, is believed to be an important factor in the pathogenesis of cervical ossification of the posterior longitudinal ligament(C-OPLL). So this research was to identify the relation between the serum leptin and bone metabolic markers and how the leptin induced osteogenic effect in C-OPLL. METHODS: Sixty-four samples were selected to determine the concentration of leptin, insulin, and alkaline phosphatase. And the association of leptin with these factors was also examined. We also evaluate the effect of leptin on the development of C-OPLL and further explored the possible underlying mechanism in vitro. RESULTS: We found that serum leptin concentrations were higher in females than in males. Serum leptin and ALP concentrations were increased significantly in C-OPLL females compared to non-OPLL females. In OPLL subjects, the serum leptin concentration corrected for body mass index correlated negatively with the ALP concentrations. In C-OPLL cells, leptin treatment led to a significant increase in mRNA expressions of ALP and OCN and formation of mineralized nodule. Our experiments reported here that osteogenic effect of leptin in C-OPLL cells could be mediated via ERK1/2, p38 MAPK, and/or JNK signaling pathways. CONCLUSIONS: From this research, we got that leptin treatment led to a significant increase in mRNA expressions of ALP and OCN and formation of mineralized nodule. And the osteogenic effect of leptin in C-OPLL cells could be mediated via ERK1/2, p38 MAPK, and/or JNK signaling pathways.

The microRNA-10a/ID3/RUNX2 axis modulates the development of Ossification of Posterior Longitudinal Ligament.

Ossification of the posterior longitudinal ligament (OPLL) presents as pathological heterotopic ossification of the spinal ligaments. However, its underlying molecular mechanism is still unclear. Our previous findings suggested that altered microRNA regulatory network are critical for the development of OPLL. Here, we set out to unveiling the detailed mechanism of those altered OPLL-specific microRNAs. We screened a set of differentially expressed OPLL-specific microRNAs from the previous sequencing data and showed that microRNA-10a actively modulates the ossification of posterior ligament cells in vitro. Using a tissue-engineered scaffold grown from 4-week-old BALB/c homozygous nude mice, we found that altered microRNA-10a expression in posterior ligament cells indeed affected the heterotopic bone formation in vivo. Furthermore, computational analysis showed that the negative ossification regulator ID3 is a functional target gene of microRNA-10a, and its expression was also significantly altered during microRNA-10a modulation both in vitro and in vivo. Also, we have demonstrated that the ossification promoting function of microRNA-10a requires ID3, as ID3 actively inhibits RUNX2. Thus, we identified a critical role for highly altered OPLL-specific microRNA-10a in regulating the development of OPLL by modulating the ID3/RUNX2 axis.

Connexin 43 Affects Osteogenic Differentiation of the Posterior Longitudinal Ligament Cells via Regulation of ERK Activity by Stabilizing Runx2 in Ossification.

AIMS: Connexin 43 is one of the most potent gap junction proteins related to osteoblast differentiation and bone formation. We hypothesized that Connexin 43 is a significant factor in osteogenic differentiation in the posterior longitudinal ligament through the regulation of extracellular signal-regulated kinases (ERK) activity by converging on Runt-related transcription factor 2 (Runx2) activity. In this study, we mapped the activity of Connexin 43 to ERK and Runx2 by extracting longitudinal ligament cell for culture and silencing Connexin expression in addition to dexamethasone treatment in vitro. METHODS: qRT-PCR, Western Blot, and Runx2-responsive Luciferase Reporter Assay were performed to detect the activity of ERK, Runx2 and the expression levels of osseous genes under Connexin 43 modification. RESULTS: Downregulation of Connexin 43 resulted in suppression of dexamethasone-induced osteogenic differentiation, inhibition of the ERK and Runx2 activity, and reduction of osseous gene expression. CONCLUSION: these data support that Connexin 43 significantly regulates osteogenic differentiation in the cells from posterior longitudinal ligament by altering the activity of ERK, and subsequently causing the modification of Runx2.

Importance of IL-6, MMP-1, IGF-1, and BAX Levels in Lumbar Herniated Disks and Posterior Longitudinal Ligament in Patients with Sciatic Pain.

PURPOSE: The aim of this study was to evaluate prognostic importance of interleukin-6 (IL-6), matrix metalloproteinase (MMP)-1, insulin-like growth factor (IGF)-1, and Bcl-2-associated X protein (BAX) levels in biopsy specimens taken from the intervertebral disk specimens and the posterior longitudinal ligaments of patients with sciatic pain. METHODS: The specimens of the intervertebral disk and the posterior longitudinal ligament were obtained from 52 patients undergoing herniectomy and diskectomy at the Neurosurgery Department of the Abant Izzet Baysal University Izzet Baysal Training and Research Hospital between April 2012 and February 2014. The immunohistochemical expressions of IL-6, MMP-1, IGF-1, and BAX were evaluated in three categories: mild, moderate, and intense. RESULTS: The IL-6 expression in the intervertebral disk specimens was intense in the sequestration group when compared with that of the "protrusion" and "extrusion" groups. The intervertebral disk specimens in "extrusion" and "sequestration" groups were stained intensely for MMP-1. The IGF-1 expression was stained intensely in the intervertebral disk tissue of the extrude group patients. For the "extrusion" and "sequestration" groups, the intervertebral disk specimens were stained intensely for BAX compared with the protrude group. The IL-6 expression in the posterior longitudinal ligament specimens was more intense in the "sequestration" and "extrusion" groups when compared with that of the protrude group. The MMP-1 expressions were milder in the sequestration group when compared with that of the "extrusion" and "protrusion" groups. CONCLUSIONS: Our findings suggest that the cytokines, enzymes, growth factors, and proapoptotic proteins, such as IL-6, MMP-1, IGF-1, and BAX, may be critical factors in the pathophysiology of the degeneration of the intervertebral disks in patients with symptomatic degenerative disk disease.

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.

Inhibitory effect of YQHYRJ recipe on osteoblast differentiation induced by BMP-2 in fibroblasts from posterior longitudinal ligament of mice.

Ossification of posterior longitudinal ligament (OPLL) is a common disease in Asian countries. Osteoblast differentiation in posterior longitudinal ligamentous fibroblast is a pathologic basis of OPLL. Nowadays, an effective pharmacotherapy for OPLL is still hunted for. YQHYRJ Recipe (YQHYRJ) is designed based on traditional Chinese medicine (TCM) theories, and previous clinic trials reported its effect on relieving syndromes of cervical spondylopathy. To clarify the YQHYRJ effect of OPLL on a cellular level, we induced mice fibroblasts from posterior longitudinal ligaments to differentiate into osteoblasts by human recombinant BMP-2, and treated them with YQHYRJ and its three sub-compounds: YQ, HY and RJ. YQHYRJ and the sub-compounds reduced the increase of fibroblast proliferation, mineralization, type I collagen secretion induced by BMP-2 via MTT, alizarin red staining and immunochemical examination. Moreover, these agents inhibited BMP-2 induced upregulation of ossification-related genes ALP, Col I and OC as well as BMP signal molecules Smad1, Smad 5 and Runx2 mRNA expression. These results suggested YQHYRJ to be effective in inhibiting osteoblast differentiation induced by BMP-2 in fibroblasts from posterior longitudinal ligament. YQHYRJ might be a promising medicine for preventing OPLL disease.

Quantitative analysis of cyclooxygenase 2 in the posterior longitudinal ligament of cervical spondylotic myelopathy.

BACKGROUND: Cervical spondylotic myelopathy (CSM), in part, results from degeneration of the posterior longitudinal ligament (PLL), which mechanically compresses the spinal cord. Much research was done on the ossification of PLL, but not concerning the non-ossifying degeneration of cervical PLL. The degeneration of cervical PLL may be related to inflammation. The aim of this study was to elucidate the pathological features of the PLL and the role of cyclooxygenase 2 (COX-2) in the degeneration of the PLL in CSM. METHODS: A total of 23 PLL specimens were collected during surgery from patients with CSM for the histological and immunohistochemical (type II collagen and Ki-67) study. For the control group 14 cervical PLL autopsy specimens were investigated in the same manner. mRNA expression of COX-2 was quantitatively measured by real-time reverse transcription-polymerase chain reaction (RT-PCR) from 18 PLL specimens of patients with CSM and 18 PLL specimens of autopsy cases. Immunohistochemistry was used to evaluate the cellular location of COX-2 in PLL. RESULTS: A distinct amount of fibrotic area, chondrometaplastic tissue and calcification were found in the PLL of the patient group, compared with the control group. Type II collagen was apparent around chondrometaplastic cells. Ki-67 positive reaction was less than 5%. A COX-2 positive reaction was found in 9 of the patient specimens (39.1%) in which the COX-2 was released from vascular endothelial cells in the PLL. However, such reactions were not found in the control group. Real-time PCR showed that the mRNA expression level of COX-2 in the patient group was significantly higher than that in the control group (P < 0.01). CONCLUSIONS: Chondrometaplastic tissue producing type II collagen was identified as the most predominant pathological feature in the degenerative PLL. The higher expression of COX-2 might be related to degeneration of the PLL in CSM.

High glucose potentiates collagen synthesis and bone morphogenetic protein-2-induced early osteoblast gene expression in rat spinal ligament cells.

Type 2 diabetes mellitus (T2DM) is an independent risk factor for ossification of the posterior longitudinal ligament, but the mechanism is unclear. We isolated cells from rat cervical spine ligaments and studied the effects of high glucose on expression of osteoblast genes to provide insight into molecular mechanism. Using these cells, high glucose stimulated the synthesis of type I collagen and significantly potentiated expression of early osteoblast genes (Runx2; alkaline phosphatase, ALP; and osteopontin, OP) induced by bone morphogenetic protein-2 (BMP-2). Notably, these effects of high glucose were fully mimicked and augmented by H(2)O(2), although blocked by the reactive oxygen species inhibitor N-acetyl cysteine. Furthermore, exposure of these cells to high glucose significantly suppressed the phosphorylation of p38MAPK while enhancing the phosphorylation of protein kinase C (PKC) in the cells. Consistent with these observations, an inhibitor of p38 augmented the potentiation of high glucose on BMP-2-induced early osteogenic gene expression, whereas the PKC inhibitor repressed the effect of high glucose on type I collagen synthesis of the cells. In conclusion, high glucose, via production of reactive oxygen species, subsequent activation of PKC, and inhibition of p38, enhances type I collagen synthesis and expression of early osteogenesis genes induced by BMP-2 in rat spinal ligament cells. Hyperglycemia may play an important role in the onset or progression of ossification of the posterior longitudinal ligament by promoting the responsiveness of ligament cells to osteogenic differentiation.

Possible role of extracellular nucleotides in ectopic ossification of human spinal ligaments.

To reveal the involvement of extracellular nucleotides in the ossification process in ossification of the posterior longitudinal ligament of the spine (OPLL), the mRNA expression profiles of P2 purinoceptors, mechanical stress-induced ATP release, and ATP-stimulated expression of osteogenic genes were analyzed in ligament cells derived from the spinal ligament of OPLL patients (OPLL cells) and non-OPLL cells derived from the spinal ligaments of cervical spondylotic myelopathy patients as a control. The extracellular ATP concentrations of OPLL cells in static culture were significantly higher than those of non-OPLL cells, and this difference was diminished in the presence of ARL67156, an ecto-nuclease inhibitor. Cyclic stretch markedly increased the extracellular ATP concentrations of both cell types to almost the same level. P2Y1 purinoceptor subtypes were intensively expressed in OPLL cells, but only weakly expressed in non-OPLL cells. Not only ATP addition but also cyclic stretch raised the mRNA levels of alkaline phosphatase and osteopontin in OPLL cells, which were blocked by MRS2179, a selective P2Y1 antagonist. These increases in the expression of osteogenic genes were not observed in non-OPLL cells. These results suggest an important role of P2Y1 and extracellular ATP in the progression of OPLL stimulated by mechanical stress.

Pathophysiological role of endothelin in ectopic ossification of human spinal ligaments induced by mechanical stress.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by progressive ectopic bone formation in the spinal ligament. To identify the genes related to ossification affected by mechanical stress during OPLL, analyses using cDNA microarray were carried out using cultured human spinal ligament cells that had been subjected to uniaxial cyclic stretching. Samples were obtained from a total of 14 patients: seven cervical or thoracic OPLL patients and seven control patients. Spinal ligament cells derived from tissues of OPLL (OPLL cells) and control (non-OPLL cells) patients were subjected to uniaxial sinusoidal cyclic stretching (0.5 Hz, 20% stretch) for various time periods (0-9 hours). cDNA microarrays revealed that ranges of distribution of both up- and downregulated genes evoked by cyclic stretching were significantly wider in OPLL cells than in non-OPLL cells. Increases in the mRNA expression of endothelin-1 (ET-1) as well as various marker genes related to ossification were also observed. mRNA expression of ET-1 and alkaline phosphatase was increased by mechanical stress in a time-dependent manner, while addition of ET-1 to static cultures of OPLL cells increased mRNA expression of alkaline phosphatase in a dose-dependent manner. During 9 hours of cyclic stretching, ET-1 release increased to about sixfold the amount observed in nonstretched cells. In non-OPLL cells, neither cyclic stretching nor ET-1 induced any increase in alkaline phosphatase expression. These results suggest that mechanical stress promotes the progression of ossification in OPLL cells through autocrine and/or paracrine mechanisms of ET-1.

Current topics in pharmacological research on bone metabolism: Promyelotic leukemia zinc finger (PLZF) and tumor necrosis factor-alpha-stimulated gene 6 (TSG-6) identified by gene expression analysis play roles in the pathogenesis of ossification of the posterior longitudinal ligament.

To understand the molecular pathogenesis of ossification of the posterior longitudinal ligament of the spine (OPLL), an ectopic bone formation disease, we performed cDNA microarray analysis on cultured ligament cells from OPLL patients to understand the molecular pathogenesis of OPLL. We identified promyelotic leukemia zinc finger (PLZF) as one of up-regulated genes and tumor necrosis factor-alpha-stimulated gene 6 (TSG-6) as one of down-regulated gene during osteoblastic differentiation. We investigated the roles of PLZF in the regulation of osteoblastic differentiation of human mesenchymal stem cells (hMSCs) and C2C12 cells. siRNA-mediated gene-silencing of PLZF resulted in a reduction of the expression of osteoblast-specific genes such as the alkaline phosphatase, collagen 1A1, Runx2/CBFA1, and osteocalcin genes in the presence of osteogenic differentiation medium (OS) in hMSCs. The overexpression of PLZF induced CBFA1 induction, suggesting that PLZF is an upstream regulator of CBFA1 and thereby participates in promoting the ossification of spinal ligament cells in OPLL patients. Adenovirus-mediated TSG-6 overexpression in hMSCs resulted in suppression of osteoblastic differentiation induced by either BMP-2 or OS. TSG-6 can bind to BMP-2 directly and thereby could inhibit BMP-2 signaling. Taken together, these findings indicate that PLZF and TSG-6 play important roles in early osteoblastic differentiation.

Immunohistochemistry of symptomatic hypertrophy of the posterior longitudinal ligament with special reference to ligamentous ossification.

STUDY DESIGN: Immnunohistochemical staining of the thickened posterior longitudinal ligament of the cervical spine. OBJECTIVES: To clarify the histological characteristics of hypertrophy of the posterior longitudinal ligament (HPLL) of the cervical spine and the relationship between HPLL and ossification of the posterior longitudinal ligament (OPLL). SETTING: Aichi Medical University, Aichi, Japan. METHODS: Eight specimens of HPLL and two of OPLL were obtained during anterior decompressive surgery on the cervical spine from patients with myelopathy. Hematoxylin and eosin staining, alcian blue staining and immunohistochemical staining with antibodies against bone morphogenetic protein (BMP), transforming growth factor (TGF)-beta, proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP) and osteopontin (OPN) were carried out on the specimens. RESULTS: HPLL showed hyalinoid degeneration, the proliferation of chondrocytes and fibroblast-like spindle cells, infiltration of vessels and small ossification. In four cases, chondroid tissue was prominent with chondrocytes, which were expressed by ALP and OPN. The cells in HPLL were weakly or moderately stained by BMP, TGF-beta and PCNA. Their expression was similar to that of OPLL. Immunohistochemical staining was negative for all cells in the control cases. CONCLUSIONS: Histological and biochemical evidence supports the hypothesis that HPLL transforms into OPLL. The positive expression of BMP and TGF-beta in HPLL cells of myelopathic patients, and their similarity to OPLL, suggest that these cells have the potential to differentiate into osteogenic cells. Of note, neither BMP nor TGF-beta was demonstrated in the PLL of control subjects. Furthermore, the expression of chondrocytes by ALP and OPN in cartilage-prominent HPLL suggests that the cartilage can be replaced by new bone.

Uni-axial cyclic stretch induces Cbfa1 expression in spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To clarify this mechanism, we investigated the effects of in vitro cyclic stretch (120% peak to peak, at 0.5 Hz) on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The mRNA expressions of Cbfa1 (an osteoblast-specific transcription factor), type I collagen, alkaline phosphatase (ALP), osteocalcin and integrin beta1 (a mechanotransducer) were increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. The effects of cyclic stretch on the spinal ligament tissues derived from OPLL and non-OPLL patients were also analyzed by immunohistochemistry using an antibody against Cbfa1. The expression of Cbfa1 was increased by cyclic stretch at the center of the spinal ligament tissues of OPLL patients, whereas no change was observed in the tissues of non-OPLL patients. Furthermore, U0126, a specific inhibitor of MAPK kinase (MEK), suppressed the stretch-induced mRNA expressions of Cbfa1, ALP and type I collagen in OPLL cells. These results suggest that in OPLL cells, mechanical stress is converted by integrin beta1 into intracellular signaling and that Cbfa1 is activated through the MAP kinase pathway. Therefore, we propose that mechanical stress plays a key role in the progression of OPLL through an increase in Cbfa1 expression.

Possible roles of CTGF/Hcs24 in the initiation and development of ossification of the posterior longitudinal ligament.

STUDY DESIGN: A biochemical and histochemical study investigating the role of CTGF/Hcs24 in the ossification of the posterior longitudinal ligament (OPLL) was conducted. OBJECTIVE: To clarify the involvement of CTGF/Hcs24 in ectopic bone formation in OPLL through endochondral ossification using human tissue. SUMMARY OF BACKGROUND DATA: Previous studies have shown that various cytokines are involved in the occurrence or development of ectopic bone formation in OPLL. Recently, the authors cloned an mRNA predominantly expressed in chondrocytes by differential display PCR and found that its gene, hcs24, is identical to that of connective tissue growth factor. It has been shown that CTGF/Hcs24 plays a major role in endochondral ossification. METHODS: Ossified ligament tissues were taken from seven male OPLL patients during surgery. Immunohistochemical staining was performed using an antibody specific for CTGF/Hcs24. Spinal ligament cells were isolated from five OPLL patients as well as five non-OPLL patients. The cells were incubated with recombinant human CTGF/Hcs24 or TGFbeta. The expression of ALP was analyzed by RT-PCR. For the effects of TGFbeta, the expression of CTGF/Hcs24 mRNA was analyzed. RESULTS: Immunohistochemical staining showed that chondrocytes in the transitional region from nonossified to ossified ligament were stained with an antibody against CTGF/Hcs24. It was found that CTGF/Hcs24 enhanced the expression ALP mRNA in OPLL cells, whereas the expression remained unchanged in non-OPLL cells. The expression of CTGF/Hcs24 mRNA in OPLL and non-OPLL cell lines was increased by TGFbeta, and there was no significant difference between the two groups. However, TGFbeta and CTGF/Hcs24 enhanced the expression of ALP mRNA only in OPLL cells. CONCLUSIONS: According to the study results, CTGF/Hcs24 may not only be an important factor in the development of endochondral ossification in OPLL, but may also be responsible for initiating osteogenesis in spinal ligament cells.

In vitro characteristics of cultured posterior longitudinal ligament tissue.

STUDY DESIGN: To determine the osteogenicity of posterior longitudinal ligament ossification, the posterior longitudinal ligament obtained during anterior cervical surgery from patients with the disorder was analyzed with in vitro cultures. OBJECTIVES: To determine the osteogenicity of the posterior longitudinal ligament. SUMMARY OF BACKGROUND DATA: The osteogenicity of posterior longitudinal ligament ossification in North America requires better documentation. METHODS: The posterior longitudinal ligament obtained during anterior cervical corpectomy with fusion from seven patients, three with ossification of the posterior longitudinal ligament documented by magnetic resonance imaging and computed tomography and four with spondylosis, was blindly submitted for in vitro culture. Explants of the posterior longitudinal ligament were placed in Dulbecco modified Eagle medium with 10% fetal calf serum, antibiotics, 4 mmol/L x L-proline, and 50 mg/L ascorbic acid. After reaching confluency, cells were trypsinized, and first-passage cells were used for all osteocalcin measurements to establish their osteoblastic phenotype. Periosteal cells, previously shown to synthesize osteocalcin, were used as a positive control. The cells were incubated with 1,25(OH)2 vitamin D3 at 10E-8 M for 72 hours in serum-free medium. The supernatants were collected and frozen, after which the quantity of osteocalcin induced by exposure to 1,25(OH)2 vitamin D3 was determined using enzyme-linked immunoassay. Control replicate cultures were measured without incubation using vitamin D3. RESULTS: Ossification of the posterior longitudinal ligament cell lines responded positively with osteocalcin synthesis in the 0.1 to 0.4 ng/M range. The cell line of the patient with spondylosis alone did not respond to vitamin D3 priming. CONCLUSIONS: Posterior longitudinal ligament cells from the three North American white patients with ossification of the posterior longitudinal ligament, when cultured in vitro, synthesized osteocalcin on vitamin D3 priming, confirming their osteoblastic phenotype, whereas posterior longitudinal ligament cells from four white patients with isolated spondylosis did not.