Ligamentum Flavum Tissue Research Articles

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

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-1β (IL-1β). 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. 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-1β, 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. The conditioned media from the isolated human LF cells naturally expressed 72-kD and 92-kD gelatinolytic activities on gelatin zymography. The IL-1β-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. TNFα and IL-1β promote proMMP-2 and proMMP-9 secretion. IL-1β 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.

Angiopoietin-like protein 2 promotes inflammatory conditions in the ligamentum flavum in the pathogenesis of lumbar spinal canal stenosis by activating interleukin-6 expression.

Chronic inflammation is thought to cause ligamentum flavum (LF) degeneration and hypertrophy in lumbar spinal canal stenosis (LSCS). Angiopoietin-like protein 2 (Angptl2) is highly expressed in hypertrophied LF. Because Angptl2 regulates interleukin-6 (IL-6) expression in various tissues, we investigated whether IL-6 is expressed in hypertrophied LF and, if so, does Angptl2 induce IL-6 expression in LF fibroblasts. LF tissue was obtained from LSCS patients and non-LSCS patients. Polymerase chain reaction (PCR) for Angptl2 and IL-6 genes and immunohistochemistry for IL-6 protein were performed in LF tissue. Fibroblasts from LF tissue were used for in vitro experiments. Expression of integrin α5β1 (an Angptl2 receptor) and Angptl2 binding to receptors on LF fibroblasts were examined by fluorescence-activated cell sorter analysis and cell adhesion assays. After Angptl2 recombinant protein treatment, NF-κB activation and IL-6 expression in LF fibroblasts were investigated by immunocytochemistry, PCR, and enzyme-linked immunosorbent assay. IL-6 mRNA expression was increased in hypertrophied LF tissue from LSCS patients and positively correlated with LF thickness and Angptl2 mRNA expression. IL-6 protein was highly expressed in LF fibroblasts in hypertrophied LF tissue. In vitro experiments demonstrated integrin α5β1 expression on LF fibroblasts and Angptl2 binding to cells via receptors. Angptl2 stimulation promoted NF-κB nuclear translocation and induced IL-6 expression and secretion in LF fibroblasts. Angptl2 promotes inflammation in LF tissue by activating IL-6 expression, leading to LF degeneration and hypertrophy.

Herniated intervertebral disk induces hypertrophy and ossification of ligamentum flavum.

In vitro experiment using degenerated human ligamentum flavum (LF) and herniated intervertebral disk (IVD). To investigate the role and effect of degenerated and herniated IVDs on LF hypertrophy and ossification. Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by aging and degenerative process. It is well known that degenerated IVDs spontaneously produce inflammatory cytokines. Therefore, we hypothesized that degenerated IVD may affect adjacent LF through secreted inflammatory cytokines. LF and herniated lumbar IVD tissues were obtained during surgical spinal procedures. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with disk supernatant from herniated IVDs. Secreted cytokines from IVD tissue culture were detected by enzyme-linked immunosorbent assay. After analysis of cytotoxicity, DNA synthesis was measured. Reverse transcription-polymerase chain reaction for mRNA expressions of types I, II, III, V, and XI collagen and osteocalcin, and histochemical stains were performed. Supernatant from tissue culture of herniated IVD showed increased production of interleukin-1α, interleukin-6, tumor necrosis factor-α, prostaglandin E2, and nitric oxide compared with disk tissue culture from traumatic condition. There was no cytotoxicity in LF cells treated with disk supernatant from herniated IVDs. There was significant increase in DNA synthesis, upregulation in mRNA expression of types III, XI collagen and osteocalcin, whereas variable expression pattern of type I and V, and strong positive stains for Von Kossa and alkaline phosphatase in LF cultures with disk supernatant. Degenerated and herniated IVDs provide an important pathomechanism in hypertrophy and ossification of the LF through inflammatory cytokines.

Angiopoietin-Like Protein 2 Induced by Mechanical Stress Accelerates Degeneration and Hypertrophy of the Ligamentum Flavum in Lumbar Spinal Canal Stenosis

Chronic inflammation and subsequent fibrosis induced by mechanical stress play an important role in ligamentum flavum (LF) hypertrophy and degeneration in patients with lumbar spinal canal stenosis (LSCS). Angiopoietin-like protein 2 (Angptl2) is a chronic inflammatory mediator induced under various pathological conditions and increases the expression of TGF-β1, which is a well-characterized mediator in LF hypertrophy. We investigated whether Angptl2 is induced by mechanical stress, and whether it contributes to LF hypertrophy and degeneration by activating the TGF-β1 signaling cascade. In this study, we investigated human LF tissue and LF fibroblasts isolated from patients who underwent lumbar surgery. We found that Angptl2 was abundantly expressed in fibroblasts of hypertrophied LF tissues at both the mRNA and protein levels. This expression was not only positively correlated with LF thickness and degeneration but also positively correlated with lumbar segmental motion. Our in vitro experiments with fibroblasts from hypertrophied LF tissue revealed that mechanical stretching stress increases the expression and secretion of Angptl2 via activation of calcineurin/NFAT pathways. In hypertrophied LF tissue, expression of TGF-β1 mRNA was also increased and TGF-β1/Smad signaling was activated. Angptl2 expression in LF tissue was positively correlated with the expression of TGF-β1 mRNA, suggesting cooperation between Angptl2 and TGF-β1 in the pathogenesis of LF hypertrophy. In vitro experiments revealed that Angptl2 increased levels of TGF-β1 and its receptors, and also activated TGF-β1/Smad signaling. Mechanical stretching stress increased TGF-β1 mRNA expression, which was partially attenuated by treatment with a calcineurin/NFAT inhibitor or Angptl2 siRNA, indicating that induction of TGF-β1 expression by mechanical stretching stress is partially mediated by Angptl2. We conclude that expression of Angptl2 induced by mechanical stress in LF fibroblasts promotes LF tissue degeneration by activation of TGF-β1/Smad signaling, which results in LF hypertrophy in patients with LSCS.

Hypertrophy of the ligamentum flavum in lumbar spinal canal stenosis is associated with increased bFGF expression

A prospective study was undertaken to investigate basic fibroblast growth factor (bFGF) expression in hypertrophic ligamentum flavum (LF) from patients with lumbar spinal canal stenosis (LSCS) and to determine whether there was a correlation of bFGF expression with LF thickness. Twenty patients with lumbar spinal canal stenosis were enrolled in this study. bFGF mRNA and protein expressions in LF were analyzed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA), respectively. The thickness of LF was measured by axial T1-weighted magnetic resonance imaging. Expression of bFGF was substantially higher in the hypertrophic LF group than in the control group (P < 0.001) as quantified by quantitative real-time PCR. In immunohistochemical study, bFGF was positively stained on the fibroblasts within hypertrophic LF compared to nonpathologic LF of controls. Subsequent ELISA analysis revealed that bFGF concentration in the hypertrophic LF group was remarkably higher than that in the control group (P = 0.003). The thickness of LF in the hypertrophic LF was significantly greater than that in the control group (P < 0.001). LSCS patients with greater severity of LF hypertrophy had significantly higher bFGF levels in the LF tissues (P < 0.001). Furthermore, the bFGF concentration exhibited a positive correlation with the LF thickness (r = 0.974, P < 0.001). These findings suggest that the increased expression of bFGF is associated with the hypertrophy of ligamentum flavum in patients with LSCS.

Inflammatory Cytokines Induce Fibrosis and Ossification of Human Ligamentum Flavum Cells

In vitro experiment using degenerated human ligamentum flavum (LF) and various inflammatory cytokines. To examine the effect of inflammatory cytokines on LF cells and to identify their roles in the pathogenesis of LF hypertrophy and ossification. Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by the degenerative processes (ie, increased collagen synthesis and chondroid metaplasia) of ligament fibroblasts. Degenerated intervertebral disk spontaneously produces inflammatory cytokines, which might affect the adjacent LF through local milieu of the spinal canal. The interlaminar portion of the LF was collected during surgical spinal procedures in 15 patients (age range, 49-78 y) with lumbar spinal stenosis. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with various inflammatory cytokines: interleukin (IL)-1α, IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO). Cytotoxicity was analyzed by MTT assays. DNA synthesis was measured with H-thymidine incorporation, and mRNA expression of types I, III, V, and XI collagen and osteocalcin were performed by reverse transcription-polymerase chain reaction. Histochemical stains such as Von Kossa were also performed to detect bone nodule formation. There was no cytotoxicity in the LF cells treated with each cytokine. There were significant increases in DNA synthesis and upregulated mRNA expression of types I, V, XI collagen and osteocalcin in LF cultures treated with various cytokines. LF cultures treated with IL-6, TNF-α, PGE2, and NO showed positive Von Kossa staining, indicating bone nodule formation from LF cells. Inflammatory cytokines (IL-6, TNF-α, PGE2, and NO) seem to play a crucial role in hypertrophy and ossification of LF. Degenerated, herniated intervertebral disks, and facet arthrosis may influence LF through inflammatory cytokines and cause hypertrophy and ossification of LF.

The effects of ligamentous injury in the human lower cervical spine

Damage is often sustained by the anterior longitudinal ligament (ALL) and ligamentum flavum (LF) in the cervical spine subsequent to whiplash or other cervical trauma. These ligaments afford substantial cervical stability when healthy, but the ability of the ALL and LF to stabilize the spine when injured is not as conclusively studied. In order to address this issue, the current study excised ALL and LF tissues from cadaveric spines and experimentally simulated whiplash-type damage to the isolated ligaments. Stiffnesses and toe region lengths were measured for both the uninjured and damaged states. These ligamentous mechanical properties were then inputted into a previously-validated finite element (FE) model of the cervical spine and the kinematic effects of various clinically relevant combinations of ligamentous injury were predicted. The data indicated three and five-fold increases in toe region length for the LF and ALL injury variants, respectively. These toe length distensions resulted in FE predictions of supra-physiologic ranges of motion, and these motions were comparable to spines with no ligamentous support. Finally, a set of cadaveric cervical spine ligament-sectioning experiments confirmed the FE predictions and supported the finding that partial injury to the relevant ligaments produces equivalent cervical kinematic signatures to spines that have completely compromised ALL and LF tissues.

Elastogenic Protein Expression of a Highly Elastic Murine Spinal Ligament: The Ligamentum Flavum

Spinal ligaments, such as the ligamentum flavum (LF), are prone to degeneration and iatrogenic injury that can lead to back pain and nerve dysfunction. Repair and regeneration strategies for these tissues are lacking, perhaps due to limited understanding of spinal ligament formation, the elaboration of its elastic fibers, maturation and homeostasis. Using immunohistochemistry and histology, we investigated murine LF elastogenesis and tissue formation from embryonic to mature postnatal stages. We characterized the spatiotemporal distribution of the key elastogenic proteins tropoelastin, fibrillin-1, fibulin-4 and lysyl oxidase. We found that elastogenesis begins in utero with the microfibril constituent fibrillin-1 staining intensely just before birth. Elastic fibers were first detected histologically at postnatal day (P) 7, the earliest stage at which tropoelastin and fibulin-4 stained intensely. From P7 to P28, elastic fibers grew in diameter and became straighter along the axis. The growth of elastic fibers coincided with intense staining of tropoelastin and fibulin-4 staining, possibly supporting a chaperone role for fibulin-4. These expression patterns correlated with reported skeletal and behavioral changes during murine development. This immunohistochemical characterization of elastogenesis of the LF will be useful for future studies investigating mechanisms for elastogenesis and developing new strategies for treatment or regeneration of spinal ligaments and other highly elastic tissues.

The Angiogenic Capacity From Ligamentum Flavum Subsequent to Inflammation

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. To test our hypothesis that activated LF, which was exposed to inflammation, induces angiogenesis, thus resulting in hypertrophy. 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. 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)-1β, IL-6, IL-8, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-β1. Naïve and macrophage-exposed LF cells that responded to TNF-α/IL-1β were compared using the same outcome measures. Hypertrophied LF tissue was stained by TGF-β1 primary antibody using immunohistochemical method. 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-1β stimulation whereas IL-6 production was increased in response to IL-1β. The coculture appeared to increase TGF-β1 secretion but the level was lower than that for macrophage-like cells alone and LF cells alone combined. LF cells interact with macrophage-like cells to produce angiogenesis-related factors except TGF-β1. 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.

The Increased Expression of Matrix Metalloproteinases Associated with Elastin Degradation and Fibrosis of the Ligamentum Flavum in Patients with Lumbar Spinal Stenosis

BackgroundOne of the characteristics of spinal stenosis is elastin degradation and fibrosis of the extracellular matrix of the ligamentum flavum. However, there have been no investigations to determine which biochemical factors cause these histologic changes. So we performed the current study to investigate the hypothesis that matrix metalloproteinases (MMPs), which possess the ability to cause extracellular matrix remodeling, may play a role as a mediator for this malady in the ligamentum flavum.MethodsThe ligamentum flavum specimens were surgically obtained from thirty patients with spinal stenosis, as well as from 30 control patients with a disc herniation. The extents of ligamentum flavum elastin degradation and fibrosis were graded (grade 0-4) with performing hematoxylin-eosin staining and Masson's trichrome staining, respectively. The localization of MMP-2 (gelatinase), MMP-3 (stromelysin) and MMP-13 (collagenase) within the ligamentum flavum tissue was determined by immunohistochemistry. The expressions of the active forms of MMP-2, MMP-3 and MMP-13 were determined by western blot analysis, and the blots were quantified using an imaging densitometer. The histologic and biochemical results were compared between the two conditions.ResultsElastin degradation and fibrosis of the ligamentum flavum were significantly more severe in the spinal stenosis samples than that in the disc herniation samples (3.14 ± 0.50 vs. 0.55 ± 0.60, p < 0.001; 3.10 ± 0.57 vs. 0.76 ± 0.52, p < 0.001, respectively). The expressions of the active form of MMPs were identified in all the ligamentum flavums of the spinal stenosis and disc herniation patients. The expressions of active MMP-2 and MMP-13 were significantly higher in the spinal stenosis samples than that in the disc herniation samples (both p < 0.05). The expression of active MMP-3 was slightly higher in the spinal stenosis samples than that in the disc herniation samples, but the difference was not statistically significant (p = 0.131). MMP-2, -3, and -13 were positively stained on the ligamentum flavum fibroblasts.ConclusionsThe current results suggest that the increased expression of active MMPs by the ligamentum flavum fibroblasts might be related to the elastin degradation and fibrosis of the ligamentum flavum in the patients who suffer with lumbar spinal stenosis.

Phenotypic Characterization of Ligamentum Flavum Cells from Patients with Ossification of Ligamentum Flavum

PurposeThe objective of this study was to determine the phenotypic characterization of ligamentum flavum cells from patients with ossification of the ligamentum flavum (OLF).Materials and MethodsLigamentum flavum tissues were harvested from OLF and non-OLF patients during surgery. OLF and non-OLF cells were isolated from explant cultures. Cultured cells were analyzed using immunofluorescence staining and reverse transcription-polymerase chain reaction.ResultsOLF cells exhibited various appearances compared with the typical fibroblast-like morphology of non-OLF cells. Expressions of collagen type I and collagen type III were observed in OLF and non-OLF cells. OLF cells uniquely expressed osteocalcin, which is a marker for osteoblasts, and collagen type II which is a marker for chondrocytes, whereas they were negative in non-OLF cells.ConclusionThese findings indicate that OLF cells have phenotypic characterization of osteoblasts and chondrocytes which could play a role in the pathophysiology of OLF.

De Novo Osteogenesis From Human Ligamentum Flavum by Adenovirus-Mediated Bone Morphogenetic Protein-2 Gene Transfer

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. 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. 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. 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. 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. 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.

HYPERTROPHY OF LIGAMENTUM FLAVUM IN LUMBAR SPINAL STENOSIS ASSOCIATED WITH INCREASED PROTEINASE INHIBITOR CONCENTRATION

Background: It is well known that age-related fibrosis, or decreases in the elastin-to-collagen ratio of the ligamentum flavum, along with hypertrophy of the ligamentum flavum, are associated with lumbar spinal stenosis. However, the molecular mechanism by which this fibrosis and hypertrophy develop is unknown. Tissue inhibitors of matrix metalloproteinase (TIMPs) are proteinase inhibitors that suppress extracellular matrix degradation. Elevated TIMP-1 and TIMP-2 expression has been implicated in various fibrotic diseases of the liver, kidney, lung, and heart. These TIMPs can also induce cellular proliferation and inhibit apoptosis in a wide range of cell types. These findings led us to postulate that TIMP-1 and TIMP-2 might also be associated with hypertrophy and fibrosis of the ligamentum flavum in lumbar spinal stenosis. Methods: We quantified and localized TIMP expression in ligamentum flavum tissues that had been obtained during surgery from thirty patients with spinal stenosis and from thirty gender-matched control patients with disc herniation. The thickness of the ligamentum flavum at the level of the facet joint was measured on axial T1-weighted magnetic resonance images. In addition, we examined ligamentum flavum tissues for the expression of markers of cellular proliferation and apoptosis. Results: The ligamentum flavum was significantly thicker in the patients with spinal stenosis (mean, 5.68 mm) than in the patients with disc herniation (mean, 2.70 mm) (p < 0.001). The concentration of TIMP-2 in the ligamentum flavum was significantly higher in the patients with spinal stenosis (mean, 12.62 ng/mL) than in those with disc herniation (mean, 8.85 ng/mL) (p = 0.028). TIMP-1 and TIMP-2 were detected in the cytoplasm of ligamentum flavum fibroblasts. TIMP-1 and TIMP-2 concentrations were associated with hypertrophy of the ligamentum flavum (p = 0.015 and p = 0.003, respectively). None of the samples from the patients with stenosis had evidence of proliferation of ligamentum flavum fibroblasts. The expression of markers for apoptosis was significantly higher in the patients with spinal stenosis (58.8%) than in those with disc herniation (26.6%) (p < 0.001). Conclusions: Increased TIMP expression has been implicated in fibrosis and hypertrophy of the extracellular matrix of several organs. Our results suggest that increased expression of TIMP-2 in ligamentum flavum fibroblasts is associated with fibrosis and hypertrophy of the ligamentum flavum in patients with spinal stenosis. Clinical Relevance: Gaining insights into the molecular pathogenesis of hypertrophy and fibrosis of the ligamentum flavum may eventually result in new therapeutic and potentially noninvasive alternatives to the treatment of spinal stenosis.

Calcification du ligament jaune du rachis cervical: À propos d’un cas et revue de la littérature

Calcification of the cervical ligamentum flavum is a rare entity observed exclusively in Japanese people. We report a new case in a 65-year-old man from Tunisia who presented with symptoms of cervical myelopathy with mild tetra paresis, sensory abnormalities and dysuria. Magnetic resonance imaging (MRI) showed a posterior compression of the spinal cord at C3-C4. CT-scan showed a calcification of the ligamentum flavum at level C3-C4, compressing the left postero-lateral aspect of the spinal cord. C3-C4 laminectomy was performed with removal of abnormal ligamentum flavum tissue. The postoperative course was uneventful and all symptoms resolved. Calcification of the cervical ligamentum flavum is a rare entity; the diagnosis is easy but the pathogenesis remains unclear. Literature regarding this pathology is reviewed.

DOES FORMALIN-FIXATION ALTER THE EXTENT OF COLLAGEN AND ELASTIN CROSSLINKS IN HUMAN SPINAL INTERVERTEBRAL DISCS AND LIGAMENTUM FLAVA?

The effect of formalin-fixation on the extent of collagen and elastin crosslinks in the matrix of human spinal intervertebral discs and ligamentum flava tissues was evaluated. Fresh discs (n = 5) and ligamentum flava (n = 22) were harvested and separated into two samples. One sample was stored in 10% buffered formalin and the other control sample in a freezer at -20°C. Using HPLC the paired anular (n = 72), nuclear (n = 22) and liagmentum flavum (n = 58) samples were dried, hydrolyzed and assayed for collagen and elastin crosslinks over a 25-week period. There were no significant differences in the collagen, isodesmosine and desmosine content, and the extent of pyridinoline and deoxypyridinoline crosslinks, between fresh and formalin-fixed disc and ligament samples after 25 weeks of formalin fixation. Short-term formalin-fixation does not significantly influence the collagen and elastin crosslink content or the extent of insoluble collagen crosslinks in spinal disc and ligamentum flavum tissues.

Osteogenesis by Transfer of Bone Morphogenetic Protein-2 cDNA in Ligamentum Flavum Cells: Approach Toward Tissue Engineering

Study Design : In-vitro experiment. Objectives : To determine the effect of bone morphogenetic protein-2 in the osteogenesis of human ligamentum flavum cells and test the feasibility of gene transfer to these cells. Summary of literature review : Bone morphogenetic protein-2 (BMP-2) is known to be an important factor in the differentiation and maintenance of the osteoblastic phenotype. Tissue engineering for osteogenesis in ligamentum flavum by BMP-2 and gene transfer has not been previously studied. Materials and Methods : Ligmentum flavum cells were harvested and cultured from surgical patients with spinal stenosis. BMP-2 was produced by transfecting pcDNA3.1/Hygro/BMP-2 into CHO cells using Lipofectamine 2000. Adenovirus-lacZ (Ad/lacZ) was also produced, and administered with BMP-2 to cell culture. The expression of lacZ was analyzed by X-gal staining. Bone formation was assessed by alkaline phosphatase, von Kossa, and alizarin Red-S staining, and the expression of osteocalcin was determined immunocytochemically. Results : Ligamentum flavum cell cultures with Ad/lacZ showed marker gene expression. BMP-2 induced osteogenesis in ligamentum flavum cells as evidenced by alkaline phosphatase, von Kosa, and alizarin Red-S staining. Also, cell culture with BMP-2 showed strong positivity with osteocalcin by immunocytochemistry. Conclusion : BMP-2 more strongly induced the osteogenesis of ligamentum flavum, and also its gene transfer to ligamentum flavum was found to be feasible. These results may open a new era of ligamentum flavum tissue engineering..