Thrombospondin-1 promotes mechanical stress-mediated ligamentum flavum hypertrophy through the TGFß1/Smad3 signaling pathway.

Lumbar spinal canal stenosis is primarily caused by ligamentum flavum hypertrophy (LFH), which is a significant pathological factor. Nevertheless, the precise molecular basis for the development of LFH remains uncertain. The current investigation observed a notable increase in thrombospondin-1 (THBS1) expression in LFH through proteomics analysis and single-cell RNA-sequencing analysis of clinical ligamentum flavum specimens. In laboratory experiments, it was demonstrated that THBS1 triggered the activation of Smad3 signaling induced by transforming growth factor ß1 (TGFß1), leading to the subsequent enhancement of COL1A2 and α-SMA, which are fibrosis markers. Furthermore, experiments conducted on a bipedal standing mouse model revealed that THBS1 played a crucial role in the development of LFH. Sestrin2 (SESN2) acted as a stress-responsive protein that suppressed the expression of THBS1, thus averting the progression of fibrosis in ligamentum flavum (LF) cells. To summarize, these results indicate that mechanical overloading causes an increase in THBS1 production, which triggers the TGFß1/Smad3 signaling pathway and ultimately results in the development of LFH. Targeting the suppression of THBS1 expression may present a novel approach for the treatment of LFH.

Quantitative evaluation of the lumbar ligamentum flavum using MRI T2-mapping: Efficacy of its clinical application in patients with lumbar spinal stenosis.

OBEJECTIVE: To perform a magnetic resonance imaging T2-mapping of the ligamentum flavum in healthy individuals and patients with lumbar spinal stenosis scheduled for surgery and compare the T2 relaxation times. SUBJECTS AND METHODS: The T2 relaxation time of the ligamentum flavum was compared among 3 groups, healthy young individuals (H group (age< 50)), healthy middle-aged and older individuals (H group (age≥50)), and patients with lumbar spinal stenosis (L group). Additionally, the thickness of the ligament was measured in the axial image plane, and the occupied area ratio of each fiber was measured by staining the surgically obtained ligament, and each was correlated with the T2 relaxation time. We also evaluated the adhesion of the ligamentum flavum with the dura mater during the surgery. RESULTS: The T2 relaxation times were significantly prolonged in H group (age ≥50) and L group (P < 0.001) compared to H group (age<50). The relationship between collagen fiber and T2 relaxation times was significantly positive (r = 0.720, P < 0.001). Moreover, the relaxation times were significantly prolonged in those with adhesion of the ligamentum flavum with the dura mater (P < 0.05). The cut-off for the relaxation time was 50 ms (sensitivity: 62.50%, false positive rate: 10.8%). CONCLUSION: Healthy middle-aged and older individuals and patients with lumbar spinal stenosis and adhesion of the ligamentum flavum with the dura mater have prolonged T2 relaxation times. Hence, the adhesion between the ligamentum flavum and dura mater should be considered in cases with a relaxation time ≥50 ms.

Safety and Efficacy Outcomes Following Spinal Endoscopic Procedures for Thoracic Ligamentous Ossification: A Systematic Review and Meta-Analysis.

STUDY DESIGN: A systematic review and meta-analysis. OBJECTIVE: This study systematically reviewed and evaluated the safety and efficacy of spinal endoscopic techniques as a treatment for thoracic ligamentum flavum ossification (TOLF). SUMMARY OF BACKGROUND DATA: The use of spinal endoscopic techniques for the treatment of TOLF has increased in recent years. The present study is the first comprehensive systematic review and meta-analysis focused on the use of spinal endoscopic techniques for TOLF. MATERIALS AND METHODS: The Cochrane Central, PubMed, Web of Science, and Embase databases were systematically searched for studies focused on patients undergoing spinal endoscopic techniques to treat symptomatic TOLF. RESULTS: This meta-analysis included 23 studies. We included 323 patients (177 males, 146 females) with a mean age of 58.40±10.06 years, with 304 total recorded lesion locations of which 245 were located in the lower thoracic spine. Complications affected 35/323 patients, and the mean operative duration for 305 patients was 108.15±47.34 minutes. For 187 patients, the mean operative bleeding was 25.13±12.54 mL, while for 87 patients the mean duration of hospitalization was 4.59±1.93 days. At last follow-up, functional assessment was performed for 260 patients, of whom 200 were in excellent condition, visual analog scale (VAS) scores were assessed for 160 patients, with a mean improvement of 4.40 (3.95, 4.86) Japanese Orthopedic Association (JOA) scores were recorded for 115 patients, with a mean improvement of 3.49 (2.79,4.18), and modified Japanese Orthopedic Association (mJOA) scores were recorded for 208 patients, with a mean improvement of 3.62 (2.89,4.35). CONCLUSIONS: These results support several advantages of spinal endoscopic techniques for the treatment of symptomatic TOLF. These include low complication rates, rapid postoperative recovery, and good functional recovery when used for single-segment, non-nodular ossification and no combined dural ossification.

ACSM5 inhibits ligamentum flavum hypertrophy by regulating lipid accumulation mediated by FABP4/PPAR signaling pathway.

BACKGROUND: Ligamentum flavum (LF) hypertrophy is the main cause of lumbar spinal canal stenosis (LSCS). Previous studies have shown that LF hypertrophy tissue exhibits abnormal lipid accumulation, but the regulatory mechanism remains unclear. The objective of this study was to explore the function and potential mechanism of ACSM5 in LF lipid accumulation. METHODS: To assess the ACSM5 expression levels, lipid accumulation and triglyceride (TG) level in LF hypertrophy and normal tissue, we utilized RT-qPCR, western blot, oil red O staining, and TG assay kit. The pearson correlation coefficient assay was used to analyze the correlation between ACSM5 levels and lipid accumulation or TG levels in LF hypertrophy tissue. The role of ACSM5 in free fatty acids (FFA)-induced lipid accumulation in LF cells was assessed in vitro, and the role of ACSM5 in LF hypertrophy in mice was verified in vivo. To investigate the underlying mechanisms of ACSM5 regulating lipid accumulation in LF, we conducted the mRNA sequencing, bioinformatics analysis, and rescue experiments. RESULTS: In this study, we found that ACSM5, which was significantly down-regulated in LF tissues, correlated with lipid accumulation. In vitro cell experiments demonstrated that overexpression of ACSM5 significantly inhibited FFA-induced lipid accumulation and fibrosis in LF cells. In vivo animal experiments further confirmed that overexpression of ACSM5 inhibited LF thickening, lipid accumulation, and fibrosis. Mechanistically, ACSM5 inhibited lipid accumulation of LF cells by inhibiting FABP4-mediated PPARγ signaling pathway, thereby improving hypertrophy and fibrosis of LF. CONCLUSIONS: our findings elucidated the important role of ACSM5 in the regulation of LF lipid accumulation and provide insight into potential therapeutic interventions for the treatment of LF hypertrophy. This study further suggested that therapeutic strategies targeting lipid deposition may be an effective potential approach to treat LF hypertrophy-induced LSCS.

Manipulation of osteogenic and adipogenic differentiation of human degenerative disc and ligamentum flavum derived progenitor cells using IL-1ß, IL-19, and IL-20.

PURPOSE: To elucidate whether pro-inflammatory cytokines might influence the commitment of intervertebral disc (IVD)- and ligamentum flavum (LF)-derived progenitor cells toward either osteogenesis or adipogenesis, specifically Interleukin-1ß (IL-1ß), IL-19, and IL-20. METHODS: Sixty patients with degenerative spondylolisthesis and lumbar or lumbosacral spinal stenosis were included in the study. Injuries to the spine, infections, and benign or malignant tumors were excluded. From nine patient samples, IVD- and LF-derived cells were isolated after primary culture, and two clinical samples were excluded due to mycoplasma infection. The effects of IL-1ß, IL-19, as well as IL-20 in regulating osteogenic and adipogenic differentiation in vitro were investigated. RESULTS: Primary IVD- and LF-derived cells were found to have a similar cell morphology and profile of surface markers (CD44, CD90, and CD105) as placenta-derived mesenchymal stem cells (MSCs). Primary IVD/LF cells have a high capacity to differentiate into osteocytes and adipocytes. IL-19 had a tendency to promote adipogenesis. IL-20 inhibited osteogenesis and promoted adipogenesis; IL-1ß promoted osteogenesis but inhibited adipogenesis. CONCLUSION: IL-1ß, IL-19, and IL-20 impact the adipogenic and osteogenic differentiation of IVD-derived and LF-derived cells. Modulating the expression of IL-1ß, IL-19, and IL-20 provides a potential avenue for controlling cell differentiation of IVD- and LF-derived cells, which might have beneficial effect for degenerative spondylolisthesis and spinal stenosis.

Decorin: a potential therapeutic candidate for ligamentum flavum hypertrophy by antagonizing TGF-ß1.

Ligamentum flavum hypertrophy (LFH) is the main physiological and pathological mechanism of lumbar spinal canal stenosis (LSCS). The specific mechanism for LFH has not been completely clarified. In this study, bioinformatic analysis, human ligamentum flavum (LF) tissues collection and analysis, and in vitro and in vivo experiments were conducted to explore the effect of decorin (DCN) on LFH pathogenesis. Here, we found that TGF-ß1, collagen I, collagen III, α-SMA and fibronectin were significantly upregulated in hypertrophic LF samples. The DCN protein expression in hypertrophic LF samples was higher than that in non-LFH samples, but the difference was not significant. DCN inhibited the expression of TGF-ß1-induced fibrosis-associated proteins in human LF cells, including collagen I, collagen III, α-SMA, and fibronectin. ELISAs showed that TGF-ß1 can upregulate PINP and PIIINP in the cell supernatant, and this effect was inhibited after DCN administration. Mechanistic studies revealed that DCN suppressed TGF-ß1-induced fibrosis by blocking the TGF-ß1/SMAD3 signaling pathway. In addition, DCN ameliorated mechanical stress-induced LFH in vivo. In summary, our findings indicated that DCN ameliorated mechanical stress-induced LFH by antagonizing the TGF-ß1/SMAD3 signaling pathway in vitro and in vivo. These findings imply that DCN is a potential therapeutic candidate for ligamentum flavum hypertrophy.

Navigation-Assisted Micro-Window Excision of Thoracic Ossification of Ligamentum Flavum (Mishima Surgery) in Professional Baseball Pitchers: A Case Report and Technical Note.

Background and Objectives: Thoracic ossification of the ligamentum flavum (OLF) often causes myelopathy and/or radiculopathy. The disease is frequently observed in East Asian populations. Although thoracic OLF in young athletes who have underwent decompression surgery has been reported, the removal of posterior spinal bony elements and ligamentous complex may often cause postoperative thoracolumbar instability. We established a novel surgical technique that preserves the posterior spinal elements, including the spinous processes, facet joints, and supraspinous and interspinous ligaments for thoracic OLF. This is the first case report to describe a navigation-assisted micro-window excision of thoracic OLF. Case: A 32-year-old male right-handed professional baseball pitcher with significant weakness and numbness in the left leg was referred to our hospital. The patient was diagnosed with thoracic OLF at T10-11 based on radiographic and magnetic resonance images in August 2022. After exposure of the left T10-11 laminae via a small unilateral incision, the location of T10-11 OLF was detected over the lamina by O-arm navigation. Then, the micro-window was made directly above the OLF using a navigated air drill, and the OLF was removed on the ipsilateral side. The contralateral side of OLF was also resected through the same micro-window, achieving complete spinal cord decompression. Results: The next day of the surgery, his leg weakness and numbness were significantly improved. Six weeks after the surgery, he started pitching. Three months after surgery, his symptoms had gone completely, and he pitched from the mound. Approximately 6 months after surgery, he successfully pitched in a professional baseball game. Conclusions: A navigation-assisted micro-window excision of thoracic OLF effectively preserved the spinal posterior bony elements and ligamentous complex. However, long-term clinical outcomes should be evaluated in future studies.

GSK-3ß and ß-Catenin Signaling Pathway is Involved in Myofibroblast Transition of Ligamentum Flavum in Lumbar Spinal Stenosis Patients.

STUDY DESIGN: Histologic analysis of the ligamentum flavum (LF) in the lumbar spine. OBJECTIVE: The objective of this study is to investigate the levels of glycogen synthase kinase-3ß (GSK-3ß) and ß-catenin in the LF tissue of patients with lumbar spinal stenosis (LSS). SUMMARY OF BACKGROUND DATA: The hypertrophy of the LF is the primary cause of the progression of LSS. Recently, Wnt signaling has been proposed as one of the molecular processes contributing to LF hypertrophy. GSK-3ß and ß-catenin are recognized to play a crucial part in the control of this signaling pathway. MATERIALS AND METHODS: From May 2020 to July 2022, LF from 51 LSS patients (LSS group) and 18 lumbar disc herniation patients (control group) were prospectively collected during surgery. Histologic analysis was investigated to confirm the progression of LF fibrosis. The levels of α-smooth muscle actin, phosphorylation of GSK-3ß (p-GSK-3ß; inactive form), and ß-catenin were analyzed in LF with Western blot analysis to reveal the GSK-3ß/ß-catenin signaling pathway. Continuous variables are expressed as mean±SD and compared using the student t test. Categorical variables are compared using the χ 2 test or Fisher exact test, as appropriate. To determine the association between p-GSK-3ß and LF thickness, the Pearson correlation coefficient was calculated based on the results of Western blot analysis. RESULTS: The LSS group was older and had thicker LF than the controls. The LSS group showed increased collagen fiber and cellularity than the controls. The levels of α-smooth muscle actin, p-GSK-3ß, and ß-catenin in the LF of the LSS group were significantly higher than that of the control group. There was a strong positive correlation between p-GSK-3ß (Ser9) level and LF thickness in LSS patients ( r =0.69, P =0.01). CONCLUSION: This research proposes a molecular mechanism for the pathogenesis of LF hypertrophy in LSS. Specifically, GSK-3ß/ß-catenin signaling appears to be related to LF hypertrophy in LSS and a positive correlation exists between p-GSK-3ß level and LF thickness. LEVEL OF EVIDENCE: Level 3.

Identification of Bone Morphometric Protein-Related Hub Genes and Construction of a Transcriptional Regulatory Network in Patients With Ossification of the Ligamentum Flavum.

STUDY DESIGN: Basic science laboratory study. OBJECTIVE: To identify hub genes related to bone morphogenetic proteins (BMPs) in the ossification of the ligamentum flavum (OLF) and analyze their functional characteristics. SUMMARY OF BACKGROUND DATA: The exact etiology and pathologic mechanism of OLF remain unclear. BMPs are pleiotropic osteoinductive proteins that may play a critical role in this condition. MATERIALS AND METHODS: The GSE106253 and GSE106256 data sets were downloaded from the Gene Expression Omnibus database. The messenger RNA (mRNA) and long noncoding RNA expression profiles were obtained from GSE106253. The microRNA expression profiles were obtained from GSE106256. Differentially expressed genes were identified between OLF and non-OLF groups and then intersected with BMP-related genes to obtain differentially expressed BMP-related genes. The least absolute shrinkage selection operator and support vector machine recursive feature elimination were used to screen hub genes. Furthermore, a competing endogenous RNA network was constructed to explain the expression regulation of the hub genes in OLF. Finally, the protein and mRNA expression levels of the hub genes were verified using Western blot and real-time polymerase chain reaction, respectively. RESULTS: We identified 671 Differentially expressed genes and 32 differentially expressed BMP-related genes. Hub genes ADIPOQ , SCD , SCX , RPS18 , WDR82 , and SPON1 , identified through the least absolute shrinkage selection operator and support vector machine recursive feature elimination analyses, showed high diagnostic values for OLF. Furthermore, the competing endogenous RNA network revealed the regulatory mechanisms of the hub genes. Real-time polymerase chain reaction showed that the mRNA expression of the hub genes was significantly downregulated in the OLF group compared with the non-OLF group. Western blot showed that the protein levels of ADIPOQ, SCD, WDR82 , and SPON1 were significantly downregulated, whereas those of SCX and RPS18 were significantly upregulated in the OLF group compared with the non-OLF group. CONCLUSION: This study is the first to identify BMP-related genes in OLF pathogenesis through bioinformatics analysis. ADIPOQ , SCD , SCX , RPS18 , WDR82 , and SPON1 were identified as hub genes for OLF. The identified genes may serve as potential therapeutic targets for treating patients with OLF.

Prevalence and distribution of ossification of the ligamenta flava in a 16th-18th century skeletal population sample from Poland.

OBJECTIVE: This study aimed to determine the prevalence of the ossification of the ligamenta flava (OLF) among skeletal remains from Poland. MATERIALS AND METHODS: 124 skeletons aged 25 years and older were analyzed. The presence and size of OLF were observed macroscopically. OLF was recorded at the cranial and caudal attachment sites of each vertebra. The following factors were analyzed: age at death, sex, and presence of other spondyloarthropathies. RESULTS: The crude prevalence of OLF in the analyzed series was 68.55 %. OLF was located most frequently in the lower thoracic spine. A statistically significant relationship was observed between the presence of OLF and age at death. OLF coincided with degenerative spondyloarthropathies of the thoracolumbar spine. CONCLUSIONS: The results of this study indicate that OLF was not a rare condition in past populations of European ancestry. Analysis of OLF prevalence in skeletal materials can contribute to reconstruction of the conditions and lifestyles of past people. SIGNIFICANCE: This study shed new light on the prevalence of OLF and provides information on the variability of OLF in past European populations. The evaluation of the prevalence of OLF represents an important contribution to the field of paleopathology in understanding disease changes in prehistoric and historic human populations. LIMITATIONS: The analyzed material came from unknown populations without demographic data. Sex and age at death were assessed using standard anthropological methods. SUGGESTIONS FOR FURTHER RESEARCH: It is important to understand the influence of sociocultural factors and physical activity patterns on the development of OLF.

Smurf1 Facilitates Oxidative Stress and Fibrosis of Ligamentum Flavum by Promoting Nrf2 Ubiquitination and Degradation.

Lumbar spinal stenosis (LSS), which can lead to irreversible neurologic damage and functional disability, is characterized by hypertrophy and fibrosis in the ligamentum flavum (LF). However, the underlying mechanism is still unclear. In the current study, the effect of Smurf1, a kind of E3 ubiquitin ligase, in promoting the fibrosis and oxidative stress of LF was investigated, and its underlying mechanism was explored. The expression of oxidative stress and fibrosis-related markers was assessed in the tissue of lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH). Next, the expression of the top 10 E3 ubiquitin ligases, obtained from Gene Expression Omnibus (GEO) dataset GSE113212, was assessed in LDH and LSS, and confirmed that Smurf1 expression was markedly upregulated in the LSS group. Furthermore, Smurf1 overexpression promotes the fibrosis and oxidative stress of LF cells. Subsequently, NRF2, an important transcription factor for oxidative stress and fibrosis, was predicted to be a target of Smurf1. Mechanistically, Smurf1 directly interacts with Nrf2 and accelerates Nrf2 ubiquitination and degradation. In conclusion, the current study suggests that Smurf1 facilitated the fibrosis and oxidative stress of LF and induced the development of LSS by promoting Nrf2 ubiquitination and degradation.

Transcriptomic alterations in hypertrophy of the ligamentum flavum: interactions of Rho GTPases, RTK, PIK3, and FGF.

PURPOSE: To analyze the differential transcriptome expression in hypertrophic ligaments flavum (HLF) compared to normal ligaments. METHODS: A case-control study was conducted that included 15 patients with hypertrophy of LF and 15 controls. Samples of LF were obtained through a lumbar laminectomy and analyzed by DNA microarrays and histology. The dysregulated biological processes, signaling pathways, and pathological markers in the HLF were identified using bioinformatics tools. RESULTS: The HLF had notable histological alterations, including hyalinosis, leukocyte infiltration, and disarrangement of collagen fibers. Transcriptomic analysis showed that up-regulated genes were associated with the signaling pathways of Rho GTPases, receptor tyrosine kinases (RTK), fibroblast growth factors (FGF), WNT, vascular endothelial growth factor, phosphoinositide 3-kinase (PIK3), mitogen-activated protein kinases, and immune system. The genes PIK3R1, RHOA, RPS27A, CDC42, VAV1, and FGF5, 9, 18, and 19 were highlighted as crucial markers in HLF. The down-expressed genes in the HLF had associations with the metabolism of RNA and proteins. CONCLUSION: Our results suggest that abnormal processes in hypertrophied LF are mediated by the interaction of the Rho GTPase, RTK, and PI3K pathways, which have not been previously described in the HLF, but for which there are currently therapeutic proposals. More studies are required to confirm the therapeutic potential of the pathways and mediators described in our results.

A muscle-preserving, spinous process-splitting approach for ossification of the ligamentum flavum in the thoracic spine in professional athletes: a report of three cases.

A muscle-preserving, spinous process-splitting approach may be a less invasive approach to conventional laminectomy in patients with thoracic ossification of the ligamentum flavum. Few reports have discussed the usefulness of this procedure for thoracic lesions in professional athletes who need highly active thoracic spinal function after surgery. The treatment of thoracic ossification of the ligamentum flavum using a spinous process-splitting approach in 3 professional athletes is presented. In all three cases the patients could return to play within 3 months after surgery without complications, and in two of the cases, there was no spinal deformity or local recurrence of ossification of the ligamentum flavum at the final follow-up at least 8 years after surgery. The spinous process-splitting approach could be a safe procedure for multi-level and all other forms of ossification of the ligamentum flavum and is less invasive to the paraspinal muscles, relieves back symptoms, and restores function for athletes.

Impact of oxidized LDL/LOX-1 system on ligamentum flavum hypertrophy.

BACKGROUND: Patients with lumbar spinal canal stenosis (LSS) often have peripheral arterial disease and aortic disease based on atherosclerosis. Oxidized LDL, which is clinically involved in the development of atherosclerosis, may also influence LF hypertrophy, but the function of the oxidized low-density lipoprotein (LDL)/lectin-type oxidized LDL receptor 1 (LOX-1) system in LF hypertrophy is unknown. We aimed to elucidate the potential involvement of oxidized LDL/LOX-1 system in ligamentum flavum (LF) hypertrophy. METHODS: A total of 43 samples were collected from LF tissues of the patients who underwent posterior lumbar spinal surgery. Immunohistochemistry for LOX-1 was performed using human LF samples. We treated the cells in vitro with inflammatory cytokines TNF-α and IL-1ß, oxidized LDL, and simvastatin. The expressions of LOX-1 and LF hypertrophy markers including type I collagen, Type III collagen, and COX-2 were assessed by real-time RT-PCR and immunocytochemistry. Phosphorylation of MAPKs and NF-κb was evaluated by Western blot after treatment with TNF-α, IL-1ß, oxidized LDL, and simvastatin. RESULTS: A significant weak correlation was observed between the number of positive cells of LOX-1 and cross-sectional area of LF on preoperative axial magnetic resonance imaging. In functional analysis, simvastatin treatment neutralized the oxidized LDL-mediated induction of mRNA expressions of LF hypertrophy markers. Western blot analysis showed that oxidized LDL as well as TNF-α and IL-1ß activated the signaling of MAPKs and NF-κb in LF cells, and that simvastatin treatment reduced the phosphorylation of all signaling. The TNF-α and IL-1ß treatments increased both mRNA and protein expression of LOX-1 in LF cells. CONCLUSION: We found a link between the oxidized LDL/LOX-1 system and LF hypertrophy. In addition, our in vitro analysis indicate that oxidized LDL may affect LF hypertrophy through signaling of MAPKs. Our results suggest that the oxidized LDL/LOX-1 system may be a potential therapeutic target for LSS.

Factors Predicting Poor Surgical Outcome in Patients with Thoracic Ossified Ligamentum Flavum - Analysis Of 106 Patients in a Tertiary Care Hospital in South India.

Background: Ossified ligamentum flavum (OLF) is the major cause of thoracic myelopathy in our locality. Surgical outcomes and their related factors for patients with thoracic OLF (T-OLF) remain unclear because of the few studies on this condition. Objectives: The present study aimed to examine the factors predicting poor surgical outcomes and the effectiveness of decompressive laminectomy and OLF resection in patients with T-OLF. Material and Methods: A total of 106 patients with T-OLF operated at our institute from 2007 to 2018 were included. The mJOA score was used in neurological assessment preoperatively and during the follow-up. Multiple regression analysis was conducted to know the best correlation between factors and surgical outcomes. Results: The mean mJOA score was 5.67 ± 2.13 preoperatively and 7.50 ± 2.60 postoperatively at the end of follow-up. The recovery rate was 43.29 ± 30.55%. After decompressive laminectomy, the mean mJOA score, modified Nurick score, and Ashworth's grade showed significant improvement (P < 0.001). Multiple regression analysis showed that the age of the patient, associated trauma, OLF level, tuberous type OLF, intramedullary signal change on T2WI, preoperative severity of myelopathy, pre-op mJOA score, and pre-op Nurick grade were significantly correlated with the surgical outcome (P < 0.001). No correlation was identified with the duration of symptoms, dural ossification, dural tear, and CSF leak (P > 0.05). Conclusion: It is important to identify preventable risk factors for poor surgical outcomes for T-OLF. Age of the patient, associated trauma, OLF level, tuberous type OLF, intramedullary signal change on T2WI, preoperative severity of myelopathy, preoperative mJOA score, and Nurick grade were important predictors of surgical outcome in our study series.

TCF7/SNAI2/miR-4306 feedback loop promotes hypertrophy of ligamentum flavum.

BACKGROUND: Hypertrophy of ligamentum flavum (HLF) is the mainly cause of lumbar spinal stenosis (LSS), but the precise mechanism of HLF formation has not been fully elucidated. Emerging evidence indicates that transcription factor 7 (TCF7) is the key downstream functional molecule of Wnt/ß-catenin signaling, which participated in regulating multiple biological processes. However, the role and underlying mechanism of TCF7 in HLF is still unclear. METHODS: We used mRNAs sequencing analysis of human LF and subsequent confirmation with RT-qPCR, western blot and immunohistochemistry to identified the TCF7 in HLF tissues and cells. Then effect of TCF7 on HLF progression was investigated both in vitro and in vivo. Mechanically, chromatin immunoprecipitation, dual-luciferase reporter assays, and rescue experiments were used to validate the regulation of TCF7/SNAI2/miR-4306 feedback loop. RESULTS: Our results identified for first time that the TCF7 expression was obviously elevated in HLF tissues and cells compared with control, and also found that TCF7 expression had significant positive correlation with LF thickness and fibrosis score. Notably, TCF7 inhibition suppressed the hyper-proliferation and fibrosis phenotype of HLF cells in vitro and ameliorated progression of HLF in mice in vivo, whereas TCF7 overexpression promoted hyper-proliferation and fibrosis phenotype of HLF cells in vitro. Our data further revealed that TCF7 interacted with SNAI2 promoter to transactivated the SNAI2 expression, thereby promoting hyper-proliferation and fibrosis phenotype of HLF cells in vitro. Furthermore, miR-4036 negatively regulated by SNAI2 could negatively feedback regulate TCF7 expression by directly binding to TCF7 mRNA 3'-UTR, thus inhibiting the hyper-proliferation and fibrosis phenotype of HLF cells in vitro. CONCLUSIONS: Our study demonstrated that TCF7 inhibition could suppress HLF formation by modulating TCF7/SNAI2/miR-4306 feedback loop, which might be considered as a novel potential therapeutic target for HLF.

Revealing the novel autophagy-related genes for ligamentum flavum hypertrophy in patients and mice model.

Background: Fibrosis is a core pathological factor of ligamentum flavum hypertrophy (LFH) resulting in degenerative lumbar spinal stenosis. Autophagy plays a vital role in multi-organ fibrosis. However, autophagy has not been reported to be involved in the pathogenesis of LFH. Methods: The LFH microarray data set GSE113212, derived from Gene Expression Omnibus, was analyzed to obtain differentially expressed genes (DEGs). Potential autophagy-related genes (ARGs) were obtained with the human autophagy regulator database. Functional analyses including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, Gene Set Enrichment Analysis (GSEA), and Gene Set Variation Analysis (GSVA) were conducted to elucidate the underlying biological pathways of autophagy regulating LFH. Protein-protein interaction (PPI) network analyses was used to obtain hub ARGs. Using transmission electron microscopy, quantitative RT-PCR, Western blotting, and immunohistochemistry, we identified six hub ARGs in clinical specimens and bipedal standing (BS) mouse model. Results: A total of 70 potential differentially expressed ARGs were screened, including 50 up-regulated and 20 down-regulated genes. According to GO enrichment and KEGG analyses, differentially expressed ARGs were mainly enriched in autophagy-related enrichment terms and signaling pathways related to autophagy. GSEA and GSVA results revealed the potential mechanisms by demonstrating the signaling pathways and biological processes closely related to LFH. Based on PPI network analysis, 14 hub ARGs were identified. Using transmission electron microscopy, we observed the autophagy process in LF tissues for the first time. Quantitative RT-PCR, Western blotting, and immunohistochemistry results indicated that the mRNA and protein expression levels of FN1, TGFß1, NGF, and HMOX1 significantly higher both in human and mouse with LFH, while the mRNA and protein expression levels of CAT and SIRT1 were significantly decreased. Conclusion: Based on bioinformatics analysis and further experimental validation in clinical specimens and the BS mouse model, six potential ARGs including FN1, TGFß1, NGF, HMOX1, CAT, and SIRT1 were found to participate in the fibrosis process of LFH through autophagy and play an essential role in its molecular mechanism. These potential genes may serve as specific therapeutic molecular targets in the treatment of LFH.

EGF Contributes to Hypertrophy of Human Ligamentum Flavum via the TGF-ß1/Smad3 Signaling Pathway.

Background: The most common spinal disorder in elderly is lumbar spinal canal stenosis (LSCS). Previous studies showed that ligamentum flavum hypertrophy (LFH) with fibrosis as the main pathological change is one of the pathogenic factors leading to LSCS. Epidermal Growth Factor (EGF) is known to have an intimate relationship with fibrosis in various tissues. Nevertheless, currently, there are few studies regarding EGF in LFH. The effect of EGF on the development of LFH is unknown, and the underlying pathomechanism remains unclear. In this study, we investigated the role of EGF in LFH and its potential molecular mechanism. Methods: First, the expression levels of EGF, phosphorylation of EGF receptor (pEGFR), Transforming growth factor-ß1 (TGF-ß1), Phosphorylated Smad3 (pSmad3), collagen I and collagen III were examined via immunohistochemistry and Western blot in LF tissues from patients with LSCS or Non-LSCS. Second, primary LF cells were isolated from adults with normal LF thickness and were cultured with different concentrations of exogenous EGF with or without erlotinib/TGF-ß1-neutralizing antibody. Results: The results showed that EGF, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression in the LSCS group was significantly higher than that in the Non-LSCS group. Meanwhile, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression was significantly enhanced in LF cells after exogenous EGF exposure, which can be notably blocked by erlotinib. In addition, pSmad3, collagen I and collagen III protein expression was blocked by TGF-ß1-neutralizing antibody. Conclusions: EGF promotes the synthesis of collagen I and collagen III via the TGF-ß1/Smad3 signaling pathway, which eventually contributes to LFH.

Clusterin negatively modulates mechanical stress-mediated ligamentum flavum hypertrophy through TGF-ß1 signaling.

Ligamentum flavum hypertrophy (LFH) is a major cause of lumbar spinal canal stenosis (LSCS). The pathomechanisms for LFH have not been fully elucidated. Isobaric tags for relative and absolute quantitation (iTRAQ) technology, proteomics assessments of human ligamentum flavum (LF), and successive assays were performed to explore the effect of clusterin (CLU) upregulation on LFH pathogenesis. LFH samples exhibited higher cell positive rates of the CLU, TGF-ß1, α-SMA, ALK5 and p-SMAD3 proteins than non-LFH samples. Mechanical stress and TGF-ß1 initiated CLU expression in LF cells. Notably, CLU inhibited the expression of mechanical stress-stimulated and TGF-ß1-stimulated COL1A2 and α-SMA. Mechanistic studies showed that CLU inhibited mechanical stress-stimulated and TGF-ß1-induced SMAD3 activities through suppression of the phosphorylation of SMAD3 and by inhibiting its nuclear translocation by competitively binding to ALK5. PRKD3 stabilized CLU protein by inhibiting lysosomal distribution and degradation of CLU. CLU attenuated mechanical stress-induced LFH in vivo. In summary, the findings showed that CLU attenuates mechanical stress-induced LFH by modulating the TGF-ß1 pathways in vitro and in vivo. These findings imply that CLU is induced by mechanical stress and TGF-ß1 and inhibits LF fibrotic responses via negative feedback regulation of the TGF-ß1 pathway. These findings indicate that CLU is a potential treatment target for LFH.

Coexistence of flavum ligament ossification with diffuse idiopathic skeletal hyperostosis in the cervical spine: Review of literature and technical note starting from a rare case.

BACKGROUND: Cervical flavum ligament ossification (C-OLF) is very rare source of myeloradiculopathy. Less than 100 cases have been reported in modern English literature up to 2020. Association between C-OLF and Diffuse Idiopathic Skeletal Hyperostosis (DISH) at cervical level has never been described. METHODS: In this article we performed a systematic review about epidemiology, physiopathology, clinical and surgical management of C-OLF. Moreover, we research its possible association with other cervical spine ligament ossification and in particular with anterior longitudinal ligament ossification. We report a case of 73 years-old woman experiencing mild cervical myeloradiculopathy caused by C6-C7 C-OLF compression and coexistence of DISH at cervico-thoracic level. A brief technical note about intraoperative management of C-OLF has also been described. RESULT: Our research found 81 previous reported case of C-OLF. The coexistence of Posterior longitudinal ligament ossification has been reported in 21.3% of C-OLF case. Conversely, we reported the first case describing the association between DISH and C-OLF. Posterior surgical decompression is the only useful treatment providing good long-term functional outcome. Instrumentation should be tailored according to pre-operative findings. CONCLUSIONS: C-OLF is a rare source of myeloradiculopathy and it may coexists with DISH probably due to alteration in the cervical mechanical stress and tendency of bone formation in patients harboring coexistent ligament ossifications. According to our result, skip en-bloc microsurgical laminectomy is safe and less invasive method to avoid complication and to provide optimal cervical spinal cord and nerve decompression avoiding CSF-leak.