LGR5 regulates osteogenic differentiation of human thoracic ligamentum flavum cells by Wnt signalling pathway.

Thoracic ossification of the ligamentum flavum (TOLF) is ectopic ossification of the spinal ligaments. Histologically, the development of TOLF can be described as the process of endochondral ossification. However, the underlying aetiology has not been completely clarified. In this investigation, the gene expression profile associated with leucine-rich repeat-containing G-protein-coupled receptors (LGR) and Wnt signalling pathway in the thoracic ligamentum flavum cells (TLFCs) of different ossification stages was analysed via RNA sequencing. We further confirmed the significant differences in the related gene expression profile by Gene Ontology (GO) enrichment analysis. LGR5 was first identified in primary human TLFCs during osteogenic differentiation. To evaluate the effect of LGR5 on osteogenic differentiation, LGR5 has been knocked down and overexpressed in human TLFCs. We observed that the knockdown of LGR5 inhibited the activity of Wnt signalling and attenuated the potential osteogenic differentiation of TLFCs, while overexpression of LGR5 activated the Wnt signalling pathway and increased osteogenic differentiation. Our results provide important evidence for the potent positive mediatory effects of LGR5 on osteogenesis by enhancing the Wnt signalling pathway in TOLF.

Effect of teriparatide on ligamentum flavum mesenchymal stem cells isolated from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) within the spinal canal sometimes leads to severe myelopathy. Teriparatide (TPD) is a recombinant human parathyroid hormone (PTH) (1-34), which promotes osteogenesis of mesenchymal stem cells (MSCs) via PTH 1 receptor (PTH1R). Although ligamentum flavum (LF)-MSCs from patients with OPLL have a high osteogenic potency, the effect of TPD on them remains unknown. In this study, we determined PTH1R expression in LF-MSCs from patients with OPLL and investigated whether TPD promotes osteogenic differentiation in them. First, LF-MSCs were isolated from patients with OPLL and cervical spondylotic myelopathy (CSM) (controls). Cultured LF-MSCs were treated with different concentrations of TPD on days 0, 7, and 14. On day 21, osteogenic gene expression was quantified. Mineralization was measured based on optical density after Alizarin Red S staining. LF-MSCs from both groups expressed PTH1R at the same level. TPD did not enhance osteogenic gene expression and mineralization in LF-MSCs from both groups. TPD did not promote the osteogenic differentiation of LF-MSCs from patients with OPLL. Thus, it may be safe for patients with OPLL. However, further confirmation of our results with in vivo studies is necessary.

Downregulation of antidifferentiation noncoding RNA promotes chondrogenic differentiation and calcification of ligamentum flavum-derived mesenchymal stem cells.

Ligamentum flavum (LF)-derived mesenchymal stem cells (MSCs) have been implicated in the pathogenesis of calcification of the ligamentum flavum (CLF) leading to the increased presence of chondrocyte-like cells and calcium deposition in CLF; however, the mechanisms of LF-MSCs in differentiation are not defined. In this study, we investigated the role of antidifferentiation noncoding RNA (ANCR) in the differentiation of LF-MSCs. We found that ANCR was downregulated in human CLF tissues. In cultured LF-MSCs, ANCR downregulation led to decreased cell proliferation but enhanced chondrogenic differentiation and calcification. In contract, ANCR overexpression increased cell proliferation but inhibited differentiation and calcification. Mechanistically, we detected a positive correlation between ANCR and enhancer of zeste homolog 2 (EZH2) in human CLF tissues. In cultured LF-MSCs, ANCR knockdown decreased while ANCR overexpression increased EZH2 expression. In addition, physical association between ANCR and EZH2 was revealed by an RNA pull-down assay. Functionally, EZH2 overexpression prevented chondrogenic differentiation and calcification enhanced by ANCR knockdown. These findings indicated that ANCR upregulates EZH2 expression and physically binds to EZH2 in LF-MSCs to suppress chondrogenic differentiation and calcification. Therefore, downregulated ANCR contributes to increasing of chondrocyte-like cells and calcium deposition in CLF. ANCR may serve as a therapeutic target for CLF.

MiR-490-3p inhibits osteogenic differentiation in thoracic ligamentum flavum cells by targeting FOXO1.

Thoracic ossification of the ligamentum flavum (TOLF) is a rare heterotopic ossification of spinal ligaments, which is the major cause of thoracic spinal canal stenosis and myelopathy. In this study, the roles of miR-490-3p and forkhead box O1 (FOXO1) in osteogenesis of human thoracic ligamentum flavum cells were investigated. MiR-490-3p was found to be down-regulated during osteogenic differentiation of thoracic ligamentum flavum cells, while their overexpression inhibited osteogenic differentiation. In addition, the analysis of target prediction and dual luciferase reporter assays supported that miR-490-3p directly targeted FOXO1 and suppressed the expression of FOXO1. Moreover, FOXO1 knockdown was displayed to attenuate the effect of miR-490-3p inhibition. ChIP assays showed that miR-490-3p negatively regulated the interaction of FOXO1 and RUNX2. These findings suggest that miR-490-3p performs an inhibitory role in osteogenic differentiation of thoracic ligamentum flavum cells by potentially targeting FOXO1.

Locally Produced IGF-1 Promotes Hypertrophy of the Ligamentum Flavum via the mTORC1 Signaling Pathway.

BACKGROUND/AIMS: Narrowing of the lumbar spinal canal is a condition called lumbar spinal stenosis (LSS) and is a high-morbidity problem in the elderly. LSS is commonly caused by hypertrophy of the ligamentum flavum (HLF). Previous studies showed that fibrosis of the ligamentum flavum (LF) largely contributed to HLF. However, the underlying pathomechanism remains unclear. Insulin-like growth factor-1 (IGF-1) is known to have an intimate relationship with fibrosis in various tissues. Nevertheless, currently, there are few studies regarding IGF-1 in HLF. In this study, we investigated the role of IGF-1 in HLF and its potential molecular mechanism of action. METHODS: First, the IGF-1, phosphorylation of IGF-1 receptor (pIGF-1R), phosphorylation of AKT (pAKT), phosphorylation of S6(pS6), collagen I and collagen III expression levels were examined via immunohistochemistry and Western blotting in LF tissues from patients with LSS or Non-LSS. Second, primary LF cells were isolated from adults with a normal LF thickness and were cultured with different concentrations of IGF-1 with or without NVP-AEW541/rapamycin. RESULTS: The results showed that IGF-1, pIGF-1R, pAKT, pS6, collagen I and collagen III protein expression in the LSS group was significantly higher than that in the Non-LSS group. Meanwhile, pIGF-1R, pAKT, pS6, collagen I and collagen III protein expression was significantly enhanced in LF cells after IGF-1 exposure, which can be notably blocked by NVP-AEW541. In addition, pS6, collagen I and collagen III protein expression was blocked by rapamycin. CONCLUSIONS: Enhanced IGF-1 promotes the synthesis of collagen I and collagen III via the mTORC1 signaling pathway, which eventually contributes to hypertrophy of the ligamentum flavum.

The posterior epidural ligament in the thoracic region: a cadaveric and histological study.

The existence of posterior epidural ligaments (PEL) has been established in the lumbar region, but they have hitherto not been shown to exist in the thoracic vertebral column. Their identification is of clinical significance in respect to incidental durotomy and the circulation of cerebrospinal fluid (CSF). Fourteen thoracic spine sections were dissected by cutting through the intervertebral disc and separating the ligamentum flavum from the vertebra above. The dural sheath was gently retracted anteriorly to identify macroscopic connections between the ligamentum flavum and the dura. Macroscopic connections observed were dissected out, retaining some dural sheath and ligamentum flavum. Histological staining with haematoxylin and eosin and Miller's elastin stain was used to investigate cellular connections. Thoracic PELs were positively identified in 5 of the 14 cadavers (35.7%). Histology showed similarities between the thoracic and lumbar PELs. Fifteen separate PELs were identified within these five thoracic sections. The thoracic PEL has sufficient tensile strength to present a risk to the integrity of the dural sheath during surgery, and surgeons should be aware of these connections when operating on the thoracic spine. PELs may also contribute to the circulation of CSF in the spinal subarachnoid space.

Lysophosphatidic Acid Induces Ligamentum Flavum Hypertrophy Through the LPAR1/Akt Pathway.

BACKGROUND/AIMS: Hypertrophic ligamentum flavum (LF) is a major cause of lumbar spinal stenosis. Our previous work showed that high levels of lysophosphatidic acid (LPA) expression are positively correlated with LF hypertrophy. This study aimed to further unveil how LPA regulates LF hypertrophy Methods: We studied LPAR1 expression in human LF cells using PCR and western blotting. Cell viability cell cycle, apoptosis rate and molecular mechanisms were assayed in LPAR1 knockdown or overexpression LF cells. LF hypertrophy and the molecular mechanism was confirmed in human samples and in in vivo studies. RESULTS: The expression of LPA and its receptor LPAR1 is significantly higher in tissues or cells harvested from hypertrophic LF compared to healthy controls. Moreover, LPA promoted LF cell proliferation by interacting with LPAR1. This conclusion is supported by the fact that depletion or overexpression of LPAR1 changed the effect of LPA on LF cell proliferation. LPA also inhibits apoptosis in LF cells through the receptor LPAR1. Importantly, we demonstrated that the LPA-LPAR1 interaction initiated Akt phosphorylation and determined cell proliferation and apoptosis. Our in vitro findings were supported by our in vivo evidence that lyophilized LPA significantly induced LF hypertrophy via the LPAR1-Akt signaling pathway. More importantly, targeted inhibition of LPAR1 by Ki16425 with a gel sponge implant effectively reduced LPA-associated LF hypertrophy. Taken together, these data indicate that LPA binds to the receptor LPAR1 to induce LF cell proliferation and inhibit apoptosis by activating AKT signaling cascades. Targeting this signaling cascade with Ki16425 is a potential therapeutic strategy for preventing LF hypertrophy. CONCLUSION: LPA-LPAR1-Akt activation is positively correlated with the proliferation and survival of LF cells. LPAR1 could be a target for new drugs and the development of new therapeutic methods for treating LF hypertrophy.

Mesenchymal Stem Cell Levels of Human Spinal Tissues.

STUDY DESIGN: Systematic review. OBJECTIVE: The aim of this study was to investigate, quantify, compare, and compile the various mesenchymal stem cell (MSC) tissue sources within human spinal tissues to act as a compendium for clinical and research application. SUMMARY OF BACKGROUND DATA: Recent years have seen a dramatic increase in academic and clinical understanding of human MSCs. Previously limited to cells isolated from bone marrow, the past decade has illicited the characterization and isolation of human MSCs from adipose, bone marrow, synovium, muscle, periosteum, peripheral blood, umbilical cord, placenta, and numerous other tissues. As researchers explore practical applications of cells in these tissues, the absolute levels of MSCs in specific spinal tissue will be critical to guide future research. METHODS: The PubMED, MEDLINE, EMBASE, and Cochrane databases were searched for articles relating to the harvest, characterization, isolation, and quantification of human MSCs from spinal tissues. Selected articles were examined for relevant data, categorized according to type of spinal tissue, and when possible, standardized to facilitate comparisons between sites. RESULTS: Human MSC levels varied widely between spinal tissues. Yields for intervertebral disc demonstrated roughly 5% of viable cells to be positive for MSC surface markers. Cartilage endplate cells yielded 18,500 to 61,875 cells/0.8 mm thick sample of cartilage end plate. Ligamentum flavum yielded 250,000 to 500,000 cells/g of tissue. Annulus fibrosus fluorescence activated cell sorting treatment found 29% of cells positive for MSC marker Stro-1. Nucleus pulposus yielded mean tissue samples of 40,584 to 234,137 MSCs per gram of tissue. CONCLUSION: Numerous tissues within and surrounding the spine represent a consistent and reliable source for the harvest and isolation of human MSCs. Among the tissues of the spine, the annulus fibrosus and ligamentum flavum each offer considerable levels of MSCs, and may prove comparable to that of bone marrow. LEVEL OF EVIDENCE: 5.

The involvement and possible mechanism of pro-inflammatory tumor necrosis factor alpha (TNF-α) in thoracic ossification of the ligamentum flavum.

Thoracic ossification of the ligamentum flavum (TOLF) is characterized by ectopic bone formation in the ligamentum flavum and is considered to be a leading cause of thoracic spinal canal stenosis and myelopathy. However, the underlying etiology is not well understood. An iTRAQ proteomics was used to reveal the involvement of inflammation factors in TOLF. TNF-α is a pro-inflammatory cytokine implicated in the pathogenesis of many human diseases. Protein profiling analysis showed that the protein level of TNF-α increased in the ossified ligamentum flavum of TOLF, which was confirmed by western blot. The effects of TNF-α on primary ligamentum flavum cells was examined. Cell proliferation assay demonstrated that primary cells from the ossified ligamentum flavum of TOLF grew faster than the control. Flow cytometry assay indicated that the proportions of cells in S phase of cell cycle of primary cells increased after TNF-α stimulation. To address the effect of TNF-α on gene expression, primary cells were derived from ligamentum flavum of TOLF patients. Culture cells were stimulated by TNF-α. RNA was isolated and analyzed by quantitative RT-PCR. G1/S-specific proteins cyclin D1 and c-Myc were upregulated after TNF-α stimulation. On the other hand, osteoblast differentiation related genes such as Bmp2 and Osterix (Osx) were upregulated in the presence of TNF-α. TNF-α activated Osx expression in a dose-dependent manner. Interestingly, a specific mitogen-activated protein kinase ERK inhibitor U0126, but not JNK kinase inhibitor SP600125, abrogated TNF-α activation of Osx expression. This suggests that TNF-α activates Osx expression through the mitogen-activated protein kinase ERK pathway. Taken together, we provide the evidence to support that TNF-α involves in TOLF probably through regulating cell proliferation via cyclin D1 and c-Myc, and promoting osteoblast differentiation via Osx.

MiR-615-3p inhibits the osteogenic differentiation of human lumbar ligamentum flavum cells via suppression of osteogenic regulators GDF5 and FOXO1.

Ossification of the ligamentum flavum (OLF) is a disease of heterotopic ossification in spinal ligaments. The key of the OLF pathogenesis is the differentiation of fibroblasts into osteoblasts. In this study, we explored the role of miR-615-3p in the osteogenic differentiation of human LF cells. The expression of miR-615-3p was detected during the osteogenic differentiation of hFOB1.19 human osteoblasts, human BMSCs, and human LF cells. The qPCR results showed that miR-615-3p was being decreased during the osteogenic differentiation of these cell lineages. Then, both gain- and loss-function experiments, respectively performed by single-strand miR-615-3p mimic and antagomir, revealed that miR-615-3p negatively regulated the osteogenesis of hLF cells, manifested by a lighter staining degree with Alizarin Red and a decreased level of osteogenic marker genes, including alkaline phosphatase (ALP), RUNX2, osterix (ostx), osteocalcin (OCN), and osteopontin (OPN). Subsequently, our data on bioinformatic analysis, 3'-UTR luciferase activity assay, and protein level detection indicated that miR-615-3p directly targeted and suppressed the expression of FOXO1 and GDF5. Furthermore, knockdown of either FOXO1 or GDF5 could inhibit the osteogenic differentiation of hLF cells, which displayed a similar effect with the miR-615-3p mimic. In conclusion, miR-615-3p negatively regulates the osteogenic differentiation of hLF cells through post-transcriptionally suppressing osteogenic regulators GDF5 and FOXO1. It can be regarded as a potential target for human OLF therapy.

MiR-132-3p Regulates the Osteogenic Differentiation of Thoracic Ligamentum Flavum Cells by Inhibiting Multiple Osteogenesis-Related Genes.

Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6.

Notch signaling pathways in human thoracic ossification of the ligamentum flavum.

This study investigated the pathological process of Notch signaling in the osteogenesis of ligamentum flavum tissues and cells, and the associated regulatory mechanisms. Notch receptors, ligands, and target genes were identified by quantitative polymerase chain reaction (qPCR) in ligamentum flavum cells and immunohistochemistry in ligamentum flavum sections from ossification of the ligamentum flavum (OLF) patients and controls. The temporospatial expression patterns of JAG1/Notch2/HES1 in human ligamentum flavum cells during osteogenic differentiation were determined by qPCR. Lentiviral vectors for Notch2 overexpression and knockdown were constructed and transfected into ligamentum flavum cells before osteogenic differentiation to examine the function of Notch signaling pathways in the osteogenic differentiation of ligamentum flavum cells. Alkaline phosphatase, Runx2, Osterix, osteocalcin, and osteopontin mRNA levels, alkaline phosphatase activity, and Alizarin Red staining were used as indicators of osteogenic differentiation. JAG1/Notch2/HES1 mRNA levels were up-regulated in ligamentum flavum cells from OLF patients, which increased during osteogenic differentiation. Immunohistochemical analysis suggested positive Notch2 expression at the ossification front. Down-regulation of Notch2 expression decelerated osteogenic differentiation of ligamentum flavum cells, and Notch2 overexpression promoted osteogenic differentiation of ligamentum flavum cells. Expression of Runx2 and Osterix increased in a manner similar to that of Notch2 during osteogenic differentiation of ligamentum flavum cells, and Notch2 knockdown and overexpression influenced their expression levels. Notch signaling plays an important role in OLF, and Notch may affect the osteogenic differentiation of ligamentum flavum cells via interactions with Runx2 and Osterix.© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1481-1491, 2016.

Tissue transglutaminase is involved in mechanical load-induced osteogenic differentiation of human ligamentum flavum cells.

Mechanical load-induced osteogenic differentiation might be the key cellular event in the calcification and ossification of ligamentum flavum. The aim of this study was to investigate the influence of tissue transglutaminase (TGM2) on mechanical load-induced osteogenesis of ligamentum flavum cells. Human ligamentum flavum cells were obtained from 12 patients undergoing lumbar spine surgery. Osteogenic phenotypes of ligamentum flavum cells, such as alkaline phosphatase (ALP), Alizarin red-S stain, and gene expression of osteogenic makers were evaluated following the administration of mechanical load and BMP-2 treatment. The expression of TGM2 was evaluated by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) analysis. Our results showed that mechanical load in combination with BMP-2 enhanced calcium deposition and ALP activity. Mechanical load significantly increased ALP and OC gene expression on day 3, whereas BMP-2 significantly increased ALP, OPN, and Runx2 on day 7. Mechanical load significantly induced TGM2 gene expression and enzyme activity in human ligamentum flavum cells. Exogenous TGM2 increased ALP and OC gene expression; while, inhibited TG activity significantly attenuated mechanical load-induced and TGM2-induced ALP activity. In summary, mechanical load-induced TGM2 expression and enzyme activity is involved in the progression of the calcification of ligamentum flavum.

Cyclic stretch enhances apoptosis in human lumbar ligamentum flavum cells via the induction of reactive oxygen species generation.

OBJECTIVE: The lumbar ligamentum flavum (LF) is an important part of the spine to maintain the stability of the spine. In this study we aimed to examine whether mechanical force by cyclic stretch could induce apoptosis in human LF cells and investigate the underlying mechanism. METHODS: LF cells were isolated from six young patients undergoing spinal surgery and then cultured in vitro. LF cells were subjected to cyclic stretch and the poptosis was detected by flow cytometry. The level of intracellular reactive oxygen species (ROS) and caspase-9 activity were measured. RESULTS: Cyclic stretch at a frequency of 0.5 Hz with 20% elongation induced the apoptosis of human LF cells in vitro, and this was correlated with increased ROS generation and activation of caspase-9. CONCLUSION: Our study suggests that cyclic stretch-induced apoptosis in human LF cells may be mediated by ROS generation and the activation of caspase-9.

Expression of matrix metalloproteinase-2 and -9 in human ligamentum flavum cells treated with tumor necrosis factor-α and interleukin-1ß.

OBJECT: An in vitro study was performed to understand the potential roles of matrix metalloproteinase (MMP)-2 and MMP-9 in the elastin degradation of human ligamentum flavum (LF) cells via treatment with tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß). Previous studies have identified a decreased elastin to collagen ratio in hypertrophic LF. Among the extracellular matrix remodeling endopeptidases, MMP-2 and MMP-9 are known to have elastolytic activity. The hypothesis that activated LF cells exposed to inflammation would secrete MMP-2 and MMP-9, thereby resulting in elastin degradation, was examined. METHODS: To examine MMP-2 and MMP-9 expression in human LF, cells were isolated and cultured from LF tissues that were obtained during lumbar disc surgery. Isolated LF cells were equally divided into 3 flasks and subcultured. Upon cellular confluency, the LF cells were treated with TNFα, IL-1ß, or none (as a control) and incubated for 48 hours. The conditioned media were collected and assayed for MMP-2 and MMP-9 using gelatin zymography and Western blot analysis. The electrophoresis bands were compared on densitometric scans using ImageJ software. RESULTS: The conditioned media from the isolated human LF cells naturally expressed 72-kD and 92-kD gelatinolytic activities on gelatin zymography. The IL-1ß-treated LF cells presented sustained increases in the proenzyme/zymogen forms of MMP-2 and -9 (proMMP-2 and proMMP-9), and activeMMP-9 expression (p = 0.001, 0.022, and 0.036, respectively); the TNFα-treated LF cells showed the most elevated proMMP9 secretion (p = 0.006), as determined by Western blot analyses. ActiveMMP-2 expression was not observed on zymography or the Western blot analysis. CONCLUSIONS: TNFα and IL-1ß promote proMMP-2 and proMMP-9 secretion. IL-1ß appears to activate proMMP-9 in human LF cells. Based on these findings, selective MMP-9 blockers or antiinflammatory drugs could be potential treatment options for LF hypertrophy.

Evaluating Osteogenic Potential of Ligamentum Flavum Cells Cultivated in Photoresponsive Hydrogel that Incorporates Bone Morphogenetic Protein-2 for Spinal Fusion.

Regenerative medicine is increasingly important in clinical practice. Ligamentum flava (LF) are typically removed during spine-related surgeries. LF may be a source of cells for spinal fusion that is conducted using tissue engineering techniques. In this investigation, LF cells of rabbits were isolated and then characterized by flow cytometry, morphological observation, and immunofluorescence staining. The LF cells were also cultivated in polyethylene (glycol) diacrylate (PEGDA) hydrogels that incorporated bone morphogenetic protein-2 (BMP-2) growth factor, to evaluate their proliferation and secretion of ECM and differentiation in vitro. The experimental results thus obtained that the proliferation, ECM secretion, and differentiation of the PEGDA-BMP-2 group exceeded those of the PEGDA group during the period of cultivation. The mineralization and histological staining results differed similarly. A nude mice model was utilized to prove that LF cells on hydrogels could undergo osteogenic differentiation in vivo. These experimental results also revealed that the PEGDA-BMP-2 group had better osteogenic effects than the PEGDA group following a 12 weeks after transplantation. According to all of these experimental results, LF cells are a source of cells for spinal fusion and PEGDA-BMP-2 hydrogel is a candidate biomaterial for spinal fusion by tissue engineering.

The angiogenic capacity from ligamentum flavum subsequent to inflammation: a critical component of the pathomechanism of hypertrophy.

STUDY DESIGN: In vitro study about angiogenic potentiality of ligamentum flavum (LF) cells using coculture of human lumbar LF cells and activated macropage-like THP-1 cells. OBJECTIVE: To test our hypothesis that activated LF, which was exposed to inflammation, induces angiogenesis, thus resulting in hypertrophy. SUMMARY OF BACKGROUND DATA: Inflammatory reactions after mechanical stress produce fibrosis and scarring of the LF that result in hypertrophy, a major pathological feature of spinal stenosis. This study evaluated the roles of LF cells in the pathomechanism of hypertrophy, focusing on angiogenesis. METHODS: To determine their response to the inflammatory reaction, human LF cells were cocultured with phorbol myristate acetate-stimulated macrophage-like THP-1 cells. The conditioned media were assayed for tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-ß1. Naïve and macrophage-exposed LF cells that responded to TNF-α/IL-1ß were compared using the same outcome measures. Hypertrophied LF tissue was stained by TGF-ß1 primary antibody using immunohistochemical method. RESULTS: Larger quantities of IL-6, IL-8, and VEGF were secreted by cocultured cells than by macrophages alone and LF cells alone combined. Prior macrophage exposure increased the secretion of IL-8 and VEGF in response to TNF-α/IL-1ß stimulation whereas IL-6 production was increased in response to IL-1ß. The coculture appeared to increase TGF-ß1 secretion but the level was lower than that for macrophage-like cells alone and LF cells alone combined. CONCLUSION: LF cells interact with macrophage-like cells to produce angiogenesis-related factors except TGF-ß1. Activated LF cells that have been exposed to macrophage, can impact the inducement of angiogenesis-related factors, suggesting that fibrosis and scarring during inflammatory reaction is the major pathomechanism of LF hypertrophy.

Combination of Runx2 and BMP2 increases conversion of human ligamentum flavum cells into osteoblastic cells.

The conversion of fibroblasts into osteoblasts requires the activation of key signaling pathways, including the BMP pathway. Although Runx2 is known to be a component of the BMP pathway, the combination of Runx2 and BMP2 has not yet been examined with respect to the conversion of fibroblasts into osteoblasts. Here, human ligamentum flavum (LF) fibroblast- like cells from six patients were tested for their conversion into osteoblasts using adenoviruses expressing Runx2 or BMP2. The forced expression of Runx2 or BMP2 in primary cultured LF cells resulted in a variety of proliferation and differentiation behaviors. Combined treatment of BMP2 plus Runx2 resulted in better osteoblastic differentiation than treatment with either component alone. These results indicate that the Runx2 and BMP2 pathways possess both common and independent target genes. Collectively, Runx2 plus BMP2 mediated efficient conversion of fibroblast-like LF cells into osteoblast- like cells, suggesting the possible use of these components for clinical applications such as spinal fusion.

Growth/differentiation factor-5 induces osteogenic differentiation of human ligamentum flavum cells through activation of ERK1/2 and p38 MAPK.

Ossification of ligamentum flavum (OLF) is a pathological ectopic ossification in the spinal ligament, leading to spinal canal stenosis, but little was known about its pathogenesis. A previous study has found growth/differentiation factor (GDF)-5 expression at ossified sites of the ligaments from OLF patients. This study aimed to investigate the osteogenic effects of GDF-5 on cultured human ligamentum flavum cells (LFCs). LFCs were isolated from human spinal ligamentum flavum, and treated with or without recombinant human (rh) GDF-5. Alkaline phosphatase (ALP) activity was measured. Expression of osteocalcin was assessed by reverse transcriptase-PCR, Western blotting and immunofluorescence. Matrix mineralization was assessed by alizarin red staining. Activation of mitogen-activated protein kinases (MAPK) ERK1/2, p38 and JNK were detected by Western blotting. We found that rhGDF-5 treatment increased ALP activity and osteocalcin expression in a time- and dose-dependent manner, and induced mineralized nodule form. In addition, rhGDF-5 challenge mediated the ERK1/2 and p38 activation but not JNK. Inhibiting this activation pharmacologically, using U0126, a ERK1/2 inhibitor, or SB203580, a p38 inhibitor, resulted in significantly lower ALP activity and osteocalcin protein expression. The present study shows that rhGDF-5 induces osteogenic differentiation of human LFCs through activation of ERK1/2 and p38 MAPK. These findings give some new insight into the pathogenesis of OLF.

Osterix is a key target for mechanical signals in human thoracic ligament flavum cells.

Mechanical stress is considered to be an important factor in the progression of thoracic ossification of the ligament flavum (TOLF). To elucidate the mechanism underlying mechanical stress-induced TOLF, we investigated the effect of stretching on cultured flavum ligament cells derived from TOLF and non-TOLF patients. We found that the mRNA expression of alkaline phosphatase (ALP), osteocalcin, Runx2, and osterix, but not that of Dlx5 and Msx2, was significantly increased by stretching in TOLF cells. In addition, the effect seems to be finely tuned by stretching-triggered activation of distinct mitogen-activated protein kinase cascades. Specifically, a p38 specific inhibitor, SB203580, significantly inhibited stretching-induced osterix expression as well as ALP activity, whereas a specific inhibitor of ERK1/2, U0126, prevented stretching-induced Runx2 expression. We showed that overexpression of osterix resulted in a significant increase of ALP activity in TOLF cells, and osterix-specific RNAi completely abrogated the stretching-induced ALP activity, indicating that osterix plays a key role in stretching-stimulated osteogenic effect in TOLF cells. These results suggest that mechanical stress plays important roles in the progression of TOLF through induction of osteogenic differentiation of TOLF cells, and our findings support that osterix functions as a molecular link between mechanostressing and osteogenic differentiation.