Aims. CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. Methods. We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. Results. We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Conclusion. Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κβ signalling may be implicated in the mediation of this mCRP-induced state. Cite this article: Bone Joint Res 2023;12(3):189–198

Discogenic low back pain is a common cause of disability, but its pathogenesis is poorly understood. We collected 19 specimens of lumbar intervertebral discs from 17 patients with discogenic low back pain during posterior lumbar interbody fusion, 12 from physiologically ageing discs and ten from normal control discs. We investigated the histological features and assessed the immunoreactive activity of neurofilament (NF200) and neuropeptides such as substance P (SP) and vasoactive-intestinal peptide (VIP) in the nerve fibres. The distinct histological characteristic of the painful disc was the formation of a zone of vascularised granulation tissue from the nucleus pulposus to the outer part of the annulus fibrosus along the edges of the fissures. SP-, NF- and VIP-immunoreactive nerve fibres in the painful discs were more extensive than in the control discs. Growth of nerves deep into the annulus fibrosus and nucleus pulposus was observed mainly along the zone of granulation tissue in the painful discs. This suggests that the zone of granulation tissue with extensive innervation along the tears in the posterior part of the painful disc may be responsible for causing the pain of discography and of discogenic low back pain

Objectives. Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Materials and Methods. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. Results. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. Conclusion. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised. Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560–568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3

Objectives. Interleukin 18 (IL-18) is a regulatory cytokine that degrades the disc matrix. Bone morphogenetic protein-2 (BMP-2) stimulates synthesis of the disc extracellular matrix. However, the combined effects of BMP-2 and IL-18 on human intervertebral disc degeneration have not previously been reported. The aim of this study was to investigate the effects of the anabolic cytokine BMP-2 and the catabolic cytokine IL-18 on human nucleus pulposus (NP) and annulus fibrosus (AF) cells and, therefore, to identify potential therapeutic and clinical benefits of recombinant human (rh)BMP-2 in intervertebral disc degeneration. Methods. Levels of IL-18 were measured in the blood of patients with intervertebral disc degenerative disease and in control patients. Human NP and AF cells were cultured in a NP cell medium and treated with IL-18 or IL-18 plus BMP-2. mRNA levels of target genes were measured by real-time polymerase chain reaction, and protein levels of aggrecan, type II collagen, SOX6, and matrix metalloproteinase 13 (MMP13) were assessed by western blot analysis. Results. The serum level of patients (IL-18) increased significantly with the grade of IVD degeneration. There was a dramatic alteration in IL-18 level between the advanced degeneration (Grade III to V) group and the normal group (p = 0.008) Furthermore, IL-18 induced upregulation of the catabolic regulator MMP13 and downregulation of the anabolic regulators aggrecan, type II collagen, and SOX6 at 24 hours, contributing to degradation of disc matrix enzymes. However, BMP-2 antagonised the IL-18 induced upregulation of aggrecan, type II collagen, and SOX6, resulting in reversal of IL-18 mediated disc degeneration. Conclusions. BMP-2 is anti-catabolic in human NP and AF cells, and its effects are partially mediated through provocation of the catabolic effect of IL-18. These findings indicate that BMP-2 may be a unique therapeutic option for prevention and reversal of disc degeneration. Cite this article: S. Ye, B. Ju, H. Wang, K-B. Lee. Bone morphogenetic protein-2 provokes interleukin-18-induced human intervertebral disc degeneration. Bone Joint Res 2016;5:412–418. DOI: 10.1302/2046-3758.59.BJR-2016-0032.R1

1. The uptake of S. 35. labelled sodium sulphate has been studied autoradiographically in the intervertebral disc of the young rabbit. 2. The sojourn of the isotope in the tissues includes an intracellular phase of approximately twenty-four hours, followed by an extracellular phase. 3. The cells exhibiting by far the greatest affinity for the sulphate ion are the peripheral groups of cells of the nucleus pulposus, while the chondrocyte-like cells of the cartilaginous segment of the annulus fibrosus are also fairly active. The central cells of the nucleus and the fibroblasts of the outer one-third of the annulus have a much lower uptake. 4. By analogy with similar studies on hyaline cartilage, and on the basis of correlation between the alcinophilia of the tissues and the concentration of the label, both before and after hyalase digestion of the tissue, it is considered that in the young rabbit disc, as in articular cartilage, the sulphate is incorporated primarily into chondroitin sulphate. 5. The elimination of the isotope from the nucleus at twenty-four days and the persistence of the label in the annulus fibrosus at thirty-two days tends to suggest that the metabolic turnover of acid mucopolysaccharide is considerably slower in the annulus than in the nucleus

1. In this investigation one was impressed by the close relationship that exists between the constituent parts of the intervertebral discs and the surrounding supporting structures. The part that is responsible for the maintenance of a co-ordinated balance between these structures, and hence for the effective mechanism of the spine, is the nucleus pulposus. 2. The cruciate arrangement of the annulus fibrosus is related to spinal function, and the angle of intersection of consecutive laminae of the annulus fibrosus is more or less constant. 3. In the Bantu it was found that the interspinous and supraspinous ligaments differ from the classical descriptions given in anatomical text-books. 4. The movements taking place between two vertebrae are comparable to those of a rocking-horse. 5. Narrowing of a disc is a progressive process which is the result of the disturbances in the balance between the components of the force to which a disc is subjected. The process is initiated by the loss of nucleus pulposus content. 6. With the approximation of two adjacent vertebrae a disturbance of the relations of the structures in the intervertebral foramen was noted. On anatomical grounds, when a disc is narrowed it appears that the most likely structures to cause pressure on the nerve root are not the disc itself, but the superior articular processes with their overlying ligamentum flavum

Sprung back is one of the commonest causes of low back pain. Its characteristic features are described. It is caused by rupture of the posterior ligaments of the spine, including sometimes the posterior longitudinal ligament and annulus fibrosus. The manner in which it is produced and its mechanical effects are discussed in detail

Forty-one cadaveric lumbar intervertebral joints from 18 spines were flexed and fatigue loaded to simulate a vigorous day's activity. The joints were then bisected and the discs examined. Twenty-three out of 41 of the discs showed distortions in the lamellae of the annulus fibrosus and, in a few of these, complete radial fissures were found in the posterior annulus

By polarising microscope and x-ray crystallographic techniques the annulus fibrosus has been shown to consist of regularly oriented sheets of collagen fibres. These results have been interpreted in terms of an elastic mechanism whereby thrust from the nucleus causes increased girth in the annulus. It is suggested that this is accomplished by a change in the angle between the axis of the fibres in adjacent uniaxial layers of the annulus. Furthermore, the loss of elasticity of the intervertebral disc associated with age would seem to be mainly due to changes occurring in the nucleus pulposus

1. As a result of degenerative changes in the intervertebral disc, nuclear tissue often herniates through its confining structures. These lesions are common, even in children, and often lead to difficulty in diagnosis. 2. The radiological manifestations of nuclear herniations into the spongiosa of the vertebral body, through the anterior part of the annulus fibrosus, beneath the epiphysial ring, and through the posterior part of the annulus are described and illustrated. The clinical significance of these radiological appearances and their pathological basis is indicated. 3. An understanding of the significance of the radiological findings in herniation of the nucleus pulposus and a careful correlation with the clinical features of the case are necessary for accurate diagnosis

The role of antibiotics in the treatment of disc-space infection is controversial. This study assessed the tissue penetration of flucloxacillin and cephradine into the normal intervertebral disc after intravenous administration of a bolus dose of antibiotic. Twenty-five discs were removed from 12 adolescent patients having anterior spinal surgery to correct scoliosis; antibiotic had been administered between 30 minutes and four hours before operation. Despite high blood levels, no antibiotic could be detected by bioassay or by high-pressure liquid chromatography (h.p.l.c.) in any of the specimens from the nucleus pulposus or the annulus fibrosus

A series of experiments showing how posture affects the lumbar spine is reviewed. Postures which flatten (that is, flex) the lumbar spine are compared with those that preserve the lumbar lordosis. Our review shows that flexed postures have several advantages: flexion improves the transport of metabolites in the intervertebral discs, reduces the stresses on the apophyseal joints and on the posterior half of the annulus fibrosus, and gives the spine a high compressive strength. Flexion also has disadvantages: it increases the stress on the anterior annulus and increases the hydrostatic pressure in the nucleus pulposus at low load levels. The disadvantages are not of much significance and we conclude that it is mechanically and nutritionally advantageous to flatten the lumbar spine when sitting and when lifting heavy weights

The spinal stenosis syndrome is a potential hazard when congenital or developmental narrowing of the bony canal, particularly in its lateral recesses where it can be demonstrated by axial tomography, places the emergent nerve root and its blood supply at risk to further small compressive elements. When lumbar disc degeneration allows rotatory and lateral instability, posterolateral bulging of the annulus fibrosus into the root canal occurs when weight is taken on the ipsilateral lower limb. Symptoms of the spinal stenosis syndrome do not arise until the development of this instability. To relieve the symptoms, the nerve root must be freely mobilised and decompressed by full lateral decompression, with partial or total facetectomy if necessary, by enucleation of the intervertebral disc and by removal of the posterolateral portions of the annuli fibrosi

We studied 135 lumbar discs from 27 spines removed post-mortem from subjects of an average age of 31.5 years. Defects of the annulus fibrosus were classified as peripheral, circumferential or radiating; the nucleus pulposus as normal, moderately or severely degenerate. Peripheral tears were more frequent in the anterior annulus, except in the L5-S1 disc. Circumferential tears were equally distributed between the anterior and the posterior annulus. Almost all the radiating tears were in the posterior annulus, and closely related to the presence of severe nuclear degeneration. Histology suggested that peripheral tears were due to trauma rather than biochemical degradation, and that they developed independently of nuclear degeneration. The association of peripheral annular lesions with low back pain is uncertain but our study suggests that they may have a role in the pathogenesis of discogenic pain

1. The normal anatomy of the intervertebral disc of immature rabbits is described. 2. An account is given of the changes that occur after an operative incision in the ventral part of the intervertebral discs of rabbits which allowed the escape of the nucleus pulposus. The account is based on observations made on fifty-five young animals killed at intervals during the twenty-five months after operation. 3. The superficial part of the wound in the annulus heals rapidly by active fibrosis. Thereafter there is a chondrification of the ventral region of the disc, followed by ossification. A prominent bony ridge ultimately ankyloses the vertebrae adjoining the disc. 4. The site of the nucleus pulposus is eventually occupied by a dense pad of fibrocartilage. A tongue of this tissue projects into the deep median part of the wound which remains unhealed. 5. A hypothesis is submitted regarding the mechanism of rupture of the annulus fibrosus and prolapse of the nucleus pulposus in man; this hypothesis is based in part on the observations of lesions in discs not subjected to operation

We have used an experimental model employing the bent tail of rats to investigate the effects of mechanical forces on bones and joints. Mechanical strain could be applied to the bones and joints of the tail without direct surgical exposure or the application of pins and wires. The intervertebral disc showed stretched annular lamellae on the convex side, while the annulus fibrosus on the concave side was pinched between the inner corners of the vertebral epiphysis. In young rats with an active growth plate, a transverse fissure appeared at the level of the hypertrophic cell layer or the primary metaphyseal trabecular zone. Metaphyseal and epiphyseal trabeculae on the compressed side were thicker and more dense than those of the distracted part of the vertebra. In growing animals, morphometric analysis of hemiepiphyseal and hemimetaphyseal areas, and the corresponding trabecular bone density, showed significant differences between the compressed and distracted sides. No differences were observed in adult rats. We found no significant differences in osteoclast number between compressed and distracted sides in either age group. Our results provide quantitative evidence of the working of ‘Wolff’s law’. The differences in trabecular density are examples of remodelling by osteoclasts and osteoblasts; our finding of no significant difference in osteoclast numbers between the hemiepiphyses in the experimental and control groups suggests that the response of living bone to altered strain is mediated by osteoblasts

Aims

In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD.

Aims

This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD).

1. Primary lumbar vertebral instability or "pseudo-spondylolisthesis" varies from about 3 millimetres to 1·7 centimetres, and is perhaps the commonest radiological sign associated with lumbo-sacral pain after the third decade of life. It was observed in 28·6 per cent of 500 consecutive cases of lumbo-sacral pain. The next commonest cause is gross disc degeneration, which is a late result of instability. 2. The secondary instability that may accompany a nuclear prolapse or osteoarthritis is excluded from this discussion. 3. This lumbar instability is an early sign of "incipient disc degeneration," occurring before narrowing of the disc space, sclerosis of the epiphysial rings, or osteophyte formation becomes evident. The instability in the lower lumbar region is caused by incomplete radial posterior tears, usually between the fourth and fifth lumbar vertebrae; and in the upper lumbar region from anterior concentric fissures or slits between some of the lamellae of the annulus fibrosus. 4. As shown radiologically, lumbar instability is commonest between L.4-5 and is rare between L.5 and sacrum because the facets between L.5-S.1 normally face forwards and backwards and thus resist anterior sliding. 5. The usual direction of antero-posterior sliding in the case of the upper four lumbar vertebrae is posterior—that is, the upper vertebra is displaced backwards on the one immediately below it during full extension in the erect position. The displacement tends to disappear on forced flexion, which may cause anterior displacement. On the other hand, the reverse displacement may exist between the fifth lumbar vertebra and the sacrum. 6. Operative treatment by bone grafting is a last resort in carefully selected individuals. After operation the patient rests in bed for three months without rigid splinting. Bone grafting is best for a localised lesion (affecting only one disc); it is generally not advisable if more than two discs are involved. 7. The results in thirty patients treated by spinal fusions showed that 70 per cent had no pain and resumed work, l3·3 per cent had improvement and resumed work, and l6·7 per cent were worse or no better