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.

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.

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.

De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer.

STUDY DESIGN: In vitro and in vivo experiment using degenerated human ligamentum flavum (LF) and Type 5 adenovirus construct with bone morphogenetic protein-2 (BMP-2) cDNA. OBJECTIVES: To demonstrate in vitro and in vivo osteogenic effect of BMP-2 gene transfer to human LF and to propose genetically modified LF as a substitute for autogenous bone graft in spinal fusion. SUMMARY OF BACKGROUND DATA: Spinal fusion is still considered to be an important option for treating various spinal disorders. To induce solid spinal fusion, osteoinductive and/or osteoconductive agents have been widely adopted. Autogenous LF, however, has never been seriously considered as a carrier for ex vivo osteoinductive gene therapy for spinal fusion. METHODS: In vitro experiment: Degenerated human LF was harvested and cultured. Type 5 adenovirus lacZ (Ad/lacZ) and BMP-2 construct (Ad/BMP-2) were produced. LF cell cultures were then exposed to Ad/BMP-2. Expressions of osteocalcin and BMP-2 mRNA were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis was performed to detect osteocalcin protein. Alkaline phosphatase and von Kossa stains were used to detect osteogenic markers and bone nodule formation, respectively. In vivo experiment: Human LF tissues treated with Ad/lacZ, Ad/BMP-2, and saline were implanted into the subcutaneous tissue of nude mice. After 4 weeks, nude mice were radiographed and killed. Implanted LF tissues were harvested and histologically stained. RESULTS: LF cell cultures with Ad/BMP-2 revealed strong expression of BMP-2 and osteocalcin mRNA in RT-PCR and osteocalcin protein in western blot analysis. LF cell culture with saline showed baseline expression of BMP-2, osteocalcin mRNA, and osteocalcin protein, respectively. Furthermore, LF cell culture with Ad/BMP-2 demonstrated the expression of alkaline phosphatase and bone nodule formation in the aforementioned histochemical stain. LF tissues with Ad/BMP-2 revealed de novo osteogenesis in nude mice, whereas LF with Ad/lacZ or saline showed only remaining LF tissue without sign of bone formation. CONCLUSION: Human LF cells transduced with Ad/BMP-2 exhibited the expression of osteogenic phenotype and bone nodule formation. Additionally, genetically modified human LF with BMP-2 cDNA clearly demonstrated de novo osteogenesis, which supports the concept that biologically modified LF can be a substitute for autogenous bone graft in spinal fusion surgery.

Regional difference in the appearance of apoptotic cell death in the ligamentum flavum of the human cervical spine.

Ossification or calcification of the ligamentum flavum (LF) is relatively common in the middle and lower cervical, thoracic, and lumbar spine but extremely rare in the upper cervical region. This clinical fact suggests that there exist local factors promoting or preventing ossification or calcification of LF. However, little is known about the differences in the ultrastructure and cellular alterations of the LF between the different spinal levels, even in the cervical spine. With electron microscopy, we examined samples of LF collected surgically from the upper and lower cervical spine regions; we then studied the apoptotic appearance of ligament cells using a preferential labeling method. We found direct evidence of apoptosis of ligament cells in the LF. Apoptosis was more apparent in the upper region samples than in the lower region samples. The spaces around the normal fibroblasts were filled with thick collagen fibrils, but the collagen fibrils disappeared around the apoptotic bodies and thin fibrils were formed. The difference of the level of apoptosis may correlate to the ultrastructural difference of LF, and our data will benefit further investigations seeking to clarify the mechanism of various pathological conditions in the human LF.

Biologic modification of ligamentum flavum cells by marker gene transfer and recombinant human bone morphogenetic protein-2.

STUDY DESIGN: The study involves an in vitro experiment using human ligamentum flavum (LF), adenovirus lacZ construct (Ad/lacZ), and recombinant human bone morphogenetic protein-2 (BMP-2). OBJECTIVES: To demonstrate the feasibility of marker gene transfer to human LF cells and the effect of BMP-2 on the osteogenic differentiation of human LF cells. SUMMARY OF BACKGROUND DATA: BMP-2 is a widely known pivotal osteoinductive agent. Clinically and experimentally, BMP-2 has proven to be an effective in spinal fusion. Degenerated LF has only been implicated to be of pathophysiological significance in spinal stenosis. However, biologic modifications of LF to enhance osteogenesis have not been attempted previously. MATERIALS AND METHODS: Human LF and cancellous bone from the ilium were harvested from patients with lumbar spinal stenosis. LF cells and osteoblasts were isolated and cultured, and adenovirus lacZ construct (Ad/ lacZ), luciferase construct (Ad/luciferase), and BMP-2 were designed and produced. LF cell cultures were then exposed to various concentrations of Ad/lacZ (25, 50, 75, 100, 150 multiplicity of infection) and BMP-2 (50, 100, 500, 1,000, and 1,500 ng/mL). Osteoblast cultures were used as a positive control for LF culture. LF cell cultures with Ad/luciferase served as viral controls for culture with Ad/ lacZ. The transgene expression of lacZ was assessed by X-gal stain and beta-galactosidase assay. Alkaline phosphatase, Von Kossa, and Alizarin red-S stains were used to confirm osteogenic differentiation and bone nodule formation. Immunocytochemical staining was also performed to detect osteocalcin expression. RESULTS: LF cell cultures transduced with Ad/lacZ showed extensive X-gal expression and increased beta-galactosidase activity compared to viral (Ad/luciferase) and saline controls. In LF cultures treated with BMP-2, robust alkaline phosphatase expression, and bone nodule formations were observed as evidenced by positive Von Kossa and Alizarin red-S staining, and the strong expression of osteocalcin. The osteogenic response of LF cells to BMP-2 was dose dependent. CONCLUSIONS: Human LF cells were found to be susceptible to adenovirus-mediated marker gene transfer, which offers the possibility of a new range of possible genetic modifications. In human LF cells, BMP-2 was found to markedly up-regulate the expression of osteogenic phenotypes and to induce bone nodule formation. The results of this study support the notion that biologically modified LF cells, i.e., LF cells treated with BMP-2, or with adenovirus-mediated BMP-2 cDNA gene transfer, may facilitate spinal fusion.

Mechanical stretching force promotes collagen synthesis by cultured cells from human ligamentum flavum via transforming growth factor-beta1.

Although mechanical stress as a result of spinal instability is known to cause hypertrophy of the ligamentum flavum resulting in degenerative spinal canal stenosis, the mechanism of the ligament hypertrophy is not well understood. In the present study, we investigated the effect of mechanical stretching force on collagen synthesis and transforming growth factor-beta1 (TGF-beta1) production using ligament cells isolated from human ligamentum flavum in vitro. Ligamentum flavum cells (LFCs) were isolated from human ligamentum flavum obtained from patients who underwent lumbar spine surgery. The LFCs were subjected to a mechanical stretching force using a commercially available stretching device that physically deformed the cells. Collagen synthesis and TGF-beta1 production levels in the LFCs were then examined. Notable increases were observed in the gene expressions of collagen types I, III, and V in LFCs subjected to mechanical stretching force. The increase in collagen gene expression of LFCs was inhibited in the presence of anti-TGF-beta1 antibodies. Production of TGF-beta1 by the LFCs also increased significantly by the mechanical stretching force. Exogenous application of TGF-beta1 was confirmed to increase collagen synthesis of the LFCs. This data indicated that mechanical stretching force can promote TGF-beta1 production by LFCs, resulting in hypertrophy of the ligament.