Ligamentum Flavum Hypertrophy in Asymptomatic and Chronic Low Back Pain Subjects

Purpose

To examine ligamentum flavum thickness using magnetic resonance (MR) images to evaluate its association with low back pain symptoms, age, gender, lumbar level, and disc characteristics.

Materials and Methods

Sixty-three individuals were part of this IRB-approved study: twenty-seven with chronic low back pain, and thirty-six as asymptomatic. All patients underwent MR imaging and computed tomography (CT) of the lumbar spine. The MR images at the mid-disc level were captured and enlarged 800% using a bilinear interpolation size conversion algorithm that allowed for enhanced image quality. Ligamentum flavum thickness was assessed using bilateral medial and lateral measurements. Disc height at each level was measured by the least-distance measurement method in three-dimensional models created by CT images taken of the same subject. Analysis of variance and t-tests were carried out to evaluate the relationship between ligamentum flavum thickness and patient variables.

Results

Ligamentum flavum thickness was found to significantly increase with older age, lower lumbar level, and chronic low back pain (p < 0.03). No difference in ligamentum flavum thickness was observed between right and left sided measurements, or between male and female subjects. Disc height and both ligamentum flavum thickness measurements showed low to moderate correlations that reached significance (p < 0.01). Additionally, a moderate and significant correlation between disc degeneration grade and ligamentum flavum thickness does exist (p <0.001).

Conclusion

By measuring ligamentum flavum thickness on MR images at two different sites and comparing degrees of disc degeneration, we found that ligamentum flavum thickness may be closely related to the pathogenesis of pain processes in the spine.     

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

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

Laminoplasty and Laminectomy Hybrid Decompression for the Treatment of Cervical Spondylotic Myelopathy with Hypertrophic Ligamentum Flavum: A Retrospective Study

Objective

To report the outcomes of a posterior hybrid decompression protocol for the treatment of cervical spondylotic myelopathy (CSM) associated with hypertrophic ligamentum flavum (HLF).

Background

Laminoplasty is widely used in patients with CSM; however, for CSM patients with HLF, traditional laminoplasty does not include resection of a pathological ligamentum flavum.

Methods

This study retrospectively reviewed 116 CSM patients with HLF who underwent hybrid decompression with a minimum of 12 months of follow-up. The procedure consisted of reconstruction of the C4 and C6 laminae using CENTERPIECE plates with spinous process autografts, and resection of the C3, C5, and C7 laminae. Surgical outcomes were assessed using Japanese Orthopedic Association (JOA) score, recovery rate, cervical lordotic angle, cervical range of motion, spinal canal sagittal diameter, bone healing rates on both the hinge and open sides, dural sac expansion at the level of maximum compression, drift-back distance of the spinal cord, and postoperative neck pain assessed by visual analog scale.

Results

No hardware failure or restenosis was noted. Postoperative JOA score improved significantly, with a mean recovery rate of 65.3±15.5%. Mean cervical lordotic angle had decreased 4.9 degrees by 1 year after surgery (P<0.05). Preservation of cervical range of motion was satisfactory postoperatively. Bone healing rates 6 months after surgery were 100% on the hinge side and 92.2% on the open side. Satisfactory decompression was demonstrated by a significantly increased sagittal canal diameter and cross-sectional area of the dural sac together with a significant drift-back distance of the spinal cord. The dural sac was also adequately expanded at the time of the final follow-up visit.

Conclusion

Hybrid laminectomy and autograft laminoplasty decompression using Centerpiece plates may facilitate bone healing and produce a comparatively satisfactory prognosis for CSM patients with HLF.     

FGF9 can induce endochondral ossification in cranial mesenchyme

Background  

The flat bones of the skull (i.e., the frontal and parietal bones) normally form through intramembranous ossification. At these sites cranial mesenchymal cells directly differentiate into osteoblasts without the formation of a cartilage intermediate. This type of ossification is distinct from endochondral ossification, a process that involves initial formation of cartilage and later replacement by bone.     

MiR‐199b‐5p inhibits osteogenic differentiation in ligamentum flavum cells by targeting JAG1 and modulating the Notch signalling pathway

Ossification of the ligamentum flavum (OLF) is a pathology almost only reported in East Asian countries. The leading cause of OLF is thoracic spinal canal stenosis and myelopathy. In this study, the role of miR‐199b‐5p and jagged 1 (JAG1) in primary ligamentum flavum cell osteogenesis was examined. MiR‐199b‐5p was found to be down‐regulated during osteogenic differentiation in ligamentum flavum cells, while miR‐199b‐5p overexpression inhibited osteogenic differentiation. In addition, JAG1 was found to be up‐regulated during osteogenic differentiation in ligamentum flavum cells, while JAG1 knockdown via RNA interference caused an inhibition of Notch signalling and osteogenic differentiation. Moreover, target prediction analysis and dual luciferase reporter assays supported the notion that JAG1 was a direct target of miR‐199b‐5p, with miR‐199b‐5p found to down‐regulate both JAG1 and Notch. Further, JAG1 knockdown was demonstrated to block the effect of miR‐199b‐5p inhibition. These findings imply that miR‐199b‐5p performs an inhibitory role in osteogenic differentiation in ligamentum flavum cells by potentially targeting JAG1 and influencing the Notch signalling pathway.     

The PI3K pathway regulates endochondral bone growth through control of hypertrophic chondrocyte differentiation

Background  

The majority of our bones develop through the process of endochondral ossification that involves chondrocyte proliferation and hypertrophic differentiation in the cartilage growth plate. A large number of growth factors and hormones have been implicated in the regulation of growth plate biology, however, less is known about the intracellular signaling pathways involved. PI3K/Akt has been identified as a major regulator of cellular proliferation, differentiation and death in multiple cell types.     

Troponin elevation in acute ischemic stroke (TRELAS) - protocol of a prospective observational trial

Background  

Levels of the cardiac muscle regulatory protein troponin T (cTnT) are frequently elevated in patients with acute ischemic stroke and elevated cTnT predicts poor outcome and mortality. The pathomechanism of troponin release may relate to co-morbid coronary artery disease and myocardial ischemia or, alternatively, to neurogenic cardiac damage due to autonomic activation after acute ischemic stroke. Therefore, there is uncertainty about how acute ischemic stroke patients with increased cTnT levels should be managed regarding diagnostic and therapeutic workup.     

Vasculature deprivation – induced osteonecrosis of the rat femoral head as a model for therapeutic trials

Experimental Osteonecrosis  

The authors' experience with experimentally produced femoral capital osteonecrosis in rats is reviewed: incising the periosteum at the base of the neck of the femur and cutting the ligamentum teres leads to coagulation necrosis of the epiphysis. The necrotic debris is substituted by fibrous tissue concomitantly with resorption of the dead soft and hard tissues by macrophages and osteoclasts, respectively. Progressively, the formerly necrotic epiphysis is repopulated by hematopoietic-fatty tissue, and replaced by architecturally abnormal and biomechanically weak bone. The femoral heads lose their smooth-surfaced hemispherical shape in the wake of the load transfer through the hip joint such that, together with regressive changes of the joint cartilage and inflammatory-hyperplastic changes of the articular membrane, an osteoarthritis-like disorder ensues.     

The paradoxical relationship between ligamentum flavum hypertrophy and developmental lumbar spinal stenosis

Background

Ligamentum flavum (LF) hypertrophy is a common cause of lumbar spinal stenosis and is thought to be degeneration-driven. Developmental spinal stenosis (DSS) is characterized by pre-existing narrowed spinal canals and is likely a developmental problem that occurs in childhood. In these cases, the LF may demonstrate different characteristics as compared to degeneration-driven stenosis. Thus, this study aimed to investigate the relationship between histological changes of LF and canal size.

Methods

Patients who had surgical decompression for lumbar spinal stenosis were prospectively recruited and divided into three groups (critical DSS, relative DSS and non-DSS) based on previously defined anteroposterior bony spinal canal diameter measurements on MRI. The degree of disc degeneration and LF thickness were also measured from L1 to S1. Surgical LF specimens were retrieved for histological assessment of fibrotic grade and area of fibrosis.

Results

A total of 19 females and 15 males (110 LF specimens) with an overall mean age of 65.9 years (SD?±?9.8 years) were recruited. DSS was found to have a significant negative correlation (p?<?0.001) with LF thickness, its fibrotic grade and area of fibrosis (%). Non-DSS exhibited a significant positive relationship with the degree of LF fibrosis. Disc degeneration and LF thickness had no correlation with LF histology.

Conclusions

Our study is the first to definitively note that degeneration is the cause of LF fibrosis in non-DSS patients; however, in contrast, an inverse relationship exists between canal size and LF fibrosis in DSS patients, suggesting a different pathomechanism. Hence, despite a similar degree of LF thickness, DSS patients have LF with less fibrosis compared with non-DSS patients. Further investigation of the cause of LF changes in DSS is necessary to understand this relationship.

     

Genome-Wide Analyses of Gene Expression during Mouse Endochondral Ossification

Background

Endochondral ossification is a complex process involving a series of events that are initiated by the establishment of a chondrogenic template and culminate in its replacement through the coordinated activity of osteoblasts, osteoclasts and endothelial cells. Comprehensive analyses of in vivo gene expression profiles during these processes are essential to obtain a complete understanding of the regulatory mechanisms involved.

Methodology/Principal Findings

To address these issues, we completed a microarray screen of three zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs both confirmed reported data and provided novel insights into endochondral ossification. Parallel comparisons of the microdissected tibiae data set with our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the tibia.

Conclusions/Significance

These studies allow us to better understand gene expression patterns in the growth plate and endochondral bones and provide an important technical resource for comparison of gene expression in diseased or experimentally-manipulated cartilages. Ultimately, this work will help to define the genomic context in which genes are expressed in long bones and to understand physiological and pathological ossification.     

Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

Background  

Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.%) of N.     

Structure and mechanical properties of the longitudinal ligaments and ligamentum flavum of the spine

Stress-strain curves were recorded from anterior and posterior longitudinal ligaments and ligamenta flava dissected from pig lumbar spines. Ligaments were examined during extension by light microscopy, to observe crimp structure, and by X-ray diffraction, to determine collagen fibril orientations. Scanning electron microscopy (SEM) was used to examine ligaments fixed at high and low strains. Initial stages of ligament extension involvd alignment of collagen fibrils. Collagen fibrils in unstrained ligamentum flavum were much more disoriented than in the longitudinal ligaments. Thus, fibril alignment, and consequent stiffening, occurred at much higher strains than for longitudinal ligaments, allowing ligamentum flavum to exploit the extensibility of its elastin.     

Integration,heterochrony, and adaptation in pedal digits of syndactylous marsupials

Background  

Marsupial syndactyly is a curious morphology of the foot found in all species of diprotodontian and peramelemorph marsupials. It is traditionally defined as a condition in which digits II and III of the foot are bound by skin and are reduced. Past treatments of marsupial syndactyly have not considered the implications of this unique morphology for broader issues of digit development and evolution, and the ongoing debate regarding its phylogenetic meaning lacks a broad empirical basis. This study undertakes the first interdisciplinary characterisation of syndactyly, using variance/covariance matrix comparisons of morphometric measurements, locomotor indices, ossification sequences, and re-assessment of the largely anecdotal data on the phylogenetic distribution of tarsal/metatarsal articulations and "incipient syndactyly".     

Global comparative transcriptome analysis of cartilage formation in vivo

Background  

During vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations. Continued growth of the condensations and differentiation of the mesenchymal cells into chondrocytes results in the formation of cartilage templates, or anlagen, which prefigure the shape of the future bones. The chondrocytes in the anlagen further differentiate by undergoing a complex sequence of maturation and hypertrophy, and are eventually replaced by mineralized bone. Regulation of the onset of chondrogenesis is incompletely understood, and would be informed by comprehensive analyses of in vivo gene expression.     

Analysis of chick (Gallus gallus) middle ear columella formation

Background

The chick middle ear bone, the columella, provides an accessible model in which to study the tissue and molecular interactions necessary for induction and patterning of the columella, as well as associated multiple aspects of endochondral ossification. These include mesenchymal condensation, chondrogenesis, ossification of the medial footplate and shaft, and joint formation between the persistent cartilage of the extracolumella and ossified columella. Middle and external ear defects are responsible for approximately 10% of congenital hearing defects. Thus, understanding the morphogenesis and the molecular mechanisms of the formation of the middle ear is important to understanding normal and abnormal development of this essential component of the hearing apparatus.

Results

The columella, which arises from proximal ectomesenchyme of the second pharyngeal arch, is induced and patterned in a dynamic multi-step process. From the footplate, which inserts into the inner ear oval window, the shaft spans the pneumatic middle ear cavity, and the extracolumella inserts into the tympanic membrane. Through marker gene and immunolabeling analysis, we have determined the onset of each stage in the columella's development, from condensation to ossification. Significantly, a single condensation with the putative shaft and extracolumella arms already distinguishable is observed shortly before initiation of five separate chondrogenic centers within these structures. Ossification begins later, with periosteum formation in the shaft and, unexpectedly, a separate periosteum in the footplate.

Conclusions

The data presented in this study document the spatiotemporal events leading to morphogenesis of the columella and middle ear structures and provide the first gene expression data for this region. These data identify candidate genes and facilitate future functional studies and elucidation of the molecular mechanisms of columella formation.     

Cranial Osteogenesis and Suture Morphology in Xenopus laevis: A Unique Model System for Studying Craniofacial Development

Background

The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied.

Methodology/Principal Findings

We examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (μCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13.

Conclusion

A better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process.