Comparative development of thoracic intervertebral discs and intra-articular ligaments in the human, monkey, mouse, and cat

Developmental features of thoracic intervertrebral discs and their association in the adult with other vertebral structures were investigated in four species. The human anulus fibrosus, nucleus pulposus, and intra-articular ligaments were compared to those of the fetal rhesus monkey, mouse, and kitten. Photomicrographs of transverse sections of intervertebral discs document the presence of intra-articular ligaments in fetuses of these four species. Both transverse and sagittal sections of kittens were used to identify the intercapital ligament as it differentiated from the dorsal part of the intra-articular ligament. Relatively frequent dorsal herniation of the thoracic nucleus pulposus in humans may be due to the vestigial nature of the human intra-articular ligament. Quadrupeds have well-developed intra-articular ligaments, which explains anatomically the paucity of dorsal protrusions of the nucleus pulposus into the vertebral canal in the thoracic region of the cat and mouse when compared to the human. The intra-articular ligament was closely associated with the developing prenatal mammalian intervertebral disc in the four species studied, and this relationship and its surgical importance are described.     

Formation of lamellar cross bridges in the annulus fibrosus of the intervertebral disc is a consequence of vascular regression

Cross bridges are radial structures within the highly organized lamellar structure of the annulus fibrosus of the intervertebral disc that connect two or more non-consecutive lamellae. Their origin and function are unknown. During fetal development, blood vessels penetrate deep within the AF and recede during postnatal growth. We hypothesized that cross bridges are the pathways left by these receding blood vessels.Initially, the presence of cross bridges was confirmed in cadaveric human discs aged 25 and 53 years. Next, L1-L2 intervertebral discs (n = 4) from sheep ranging in age from 75 days fetal gestation to adult were processed for paraffin histology. Mid-sagittal sections were immunostained for endothelial cell marker PECAM-1. The anterior and posterior AF were imaged using differential interference contrast microscopy, and the following parameters were quantified: total number of distinct lamellae, total number of cross bridges, percentage of cross bridges staining positive for PECAM-1, cross bridge penetration depth (% total lamellae), and PECAM-1 positive cross bridge penetration depth.Cross bridges were first observed at 100 days fetal gestation. The overall number peaked in neonates then remained relatively unchanged. The percentage of PECAM-1 positive cross bridges declined progressively from almost 100% at 100 days gestation to less than 10% in adults. Cross bridge penetration depth peaked in neonates then remained unchanged at subsequent ages. Depth of PECAM-1 positive cross bridges decreased progressively after birth. Findings were similar for both the anterior and posterior.The AF lamellar architecture is established early in development. It later becomes disrupted as a consequence of vascularization. Blood vessels then recede, perhaps due to increasing mechanical stresses in the surrounding matrix. In this study we present evidence that the pathways left by receding blood vessels remain as lamellar cross bridges. It is unclear whether the presence of cross bridges in the aging and degenerating intervertebral disc would be advantageous or detrimental, and this question should be addressed by future studies.     

Immunolocalization of type X collagen in human lumbar intervertebral discs during ageing and degeneration

 Type X collagen has so far not been reported to occur in human intervertebral discs. The objective of this study was therefore to investigate the occurrence of type X collagen in human lumbar intervertebral discs during ageing and degeneration. Ninety intervertebral discs with adjacent endplates were excised in toto from individuals (0–86 years) without known spinal disease and were processed for routine decalcified histology. Appropriate slices of each disc were processed for immunohistochemistry using a type-spec ific, monoclonal antibody raised against human type X collagen. Each intervertebral disc was examined for macroscopic and histomorphological features of disc degeneration. Immunohistochemically, a positive specific type X staining was observed in the hypertrophic zone of the growth plate and only in the interstitial matrix of juvenile (<2 years) nucleus pulposus. In adult discs, type X collagen could be localized in conjunction with advanced disc degeneration and first occurred in the disc matrix (i.e., pericellular region) of a 47-year-old specimen. Positive type X staining of the disc matrix was more frequently found in senile (>70 years) discs with end stages of disc degeneration. This study provides the first evidence for the occurrence of type X collagen in human lumbar intervertebral discs and it appears that type X collagen is re-expressed in late stages of disc degeneration. Accepted: 24 April 1997     

Three-dimensional inhomogeneous triphasic finite-element analysis of physical signals and solute transport in human intervertebral disc under axial compression

A 3D inhomogeneous finite-element model for charged hydrated soft tissues containing charged/uncharged solutes was developed and applied to analyze the mechanical, chemical, and electrical signals within the human intervertebral disc during an axial unconfined compression. The effects of tissue properties and boundary conditions on the physical signals and the transport of fluid and solute were investigated. The numerical simulation showed that, during disc compression, the fluid pressurization and the effective (von Misses) solid stress were more pronounced in the annulus fibrosus (AF) region near the interface between AF and nucleus pulposus (NP). In NP, the distributions of the fluid pressure, effective stress, and electrical potential were more uniform than those in AF. The electrical signals were very sensitive to fixed charge density. Changes in material properties of NP (water content, fixed charge density, and modulus) affected fluid pressure, electrical potential, effective stress, and solute transport in the disc. This study is important for understanding disc biomechanics, disc nutrition, and disc mechanobiology.     

Temporal changes of mechanical signals and extracellular composition in human intervertebral disc during degenerative progression

In this study, a three-dimensional finite element model was used to investigate the changes in tissue composition and mechanical signals within human lumbar intervertebral disc during the degenerative progression. This model was developed based on the cell-activity coupled mechano-electrochemical mixture theory. The disc degeneration was simulated by lowering nutrition levels at disc boundaries, and the temporal and spatial distributions of the fixed charge density, water content, fluid pressure, Von Mises stress, and disc deformation were analyzed. Results showed that fixed charge density, fluid pressure, and water content decreased significantly in the nucleus pulposus (NP) and the inner to middle annulus fibrosus (AF) regions of the degenerative disc. It was found that, with degenerative progression, the Von Mises stress (relative to that at healthy state) increased within the disc, with a larger increase in the outer AF region. Both the disc volume and height decreased with the degenerative progression. The predicted results of fluid pressure change in the NP were consistent with experimental findings in the literature. The knowledge of the variations of temporal and spatial distributions of composition and mechanical signals within the human IVDs provide a better understanding of the progression of disc degeneration.     

Validation and application of an intervertebral disc finite element model utilizing independently constructed tissue-level constitutive formulations that are nonlinear,anisotropic, and time-dependent

Finite element (FE) models are advantageous in the study of intervertebral disc mechanics as the stress–strain distributions can be determined throughout the tissue and the applied loading and material properties can be controlled and modified. However, the complicated nature of the disc presents a challenge in developing an accurate and predictive disc model, which has led to limitations in FE geometry, material constitutive models and properties, and model validation. The objective of this study was to develop a new FE model of the intervertebral disc, to validate the model?s nonlinear and time-dependent responses without tuning or calibration, and to evaluate the effect of changes in nucleus pulposus (NP), cartilaginous endplate (CEP), and annulus fibrosus (AF) material properties on the disc mechanical response. The new FE disc model utilized an analytically-based geometry. The model was created from the mean shape of human L4/L5 discs, measured from high-resolution 3D MR images and averaged using signed distance functions. Structural hyperelastic constitutive models were used in conjunction with biphasic-swelling theory to obtain material properties from recent tissue tests in confined compression and uniaxial tension. The FE disc model predictions fit within the experimental range (mean±95% confidence interval) of the disc?s nonlinear response for compressive slow loading ramp, creep, and stress-relaxation simulations. Changes in NP and CEP properties affected the neutral-zone displacement but had little effect on the final stiffness during slow-ramp compression loading. These results highlight the need to validate FE models using the disc?s full nonlinear response in multiple loading scenarios.     

Aquaporin expression in the human intervertebral disc

The nucleus pulposus (NP) of the human intervertebral disc (IVD) is a hyperosmotic tissue that is subjected to daily dynamic compressive loads. In order to survive within this environment the resident chondrocyte-like cells must be able to control their cell volume, whilst also controlling the anabolism and catabolism of their extra-cellular matrix. Recent studies have demonstrated expression of a range of bi-directional, transmembrane water and solute transporters, named aquaporins (AQPs), within chondrocytes of articular cartilage. The aim of this study was to use immunohistochemsitry to investigate the expression of aquaporins 1, 2 and 3 within the human IVD. Results demonstrated expression of both AQP-1 and -3 by cells within the NP and inner annulus fibrosus (AF), while outer AF cells lacked expression of AQP-1 and showed very low numbers of AQP-3 immunopositive cells. Cells from all regions were negative for AQP-2. Therefore this study demonstrates similarities in the phenotype of NP cells and articular chondrocytes, which may be due to similarities in tissue osmolarity and mechanobiology. The decrease in expression of AQPs from the NP to the outer AF may signify changes in cellular phenotype in response to differences in mechanbiology, osmolarity and hydration between the gelatinous NP and the fibrous AF.     

Intervertebral disc viscoelastic parameters and residual mechanics spatially quantified using a hybrid confined/in situ indentation method

With advancing age, injury, musculoskeletal pathology or a combination of these, a degenerative cascade of biomechanical, biochemical, and nutritional alterations diminish the intervertebral discs' ability to maintain its structure and function. While the biomechanics of isolated disc tissues has been investigated across this degenerative spectrum, none have attempted to retain the in situ disc-endplate morphology during compressive tissue characterization. The objective of this study was to spatially quantify the viscoelastic parameters of the intervertebral disc throughout degeneration, including the as yet unreported residual stress/strain. This required the development of a hybrid confined/in situ indentation methodology, which preserves the disc structural morphology. At four locations of the disc (anterior-AF, right and left lateral AF, and NP) stress-relaxation tests were performed using the hybrid confined/in situ indentation method, which utilizes the vertebral endplate as the porous indenter tip. This method allows the endplate to remain interwoven with the disc tissue, retaining its native orientation. Healthy disc tissue exhibited significantly higher residual stress values compared to both moderate and severe degeneration in all locations (p<0.0156). Furthermore, the equilibrium stress at 15% strain (stress relaxation) was significantly diminished with advancing disc degeneration (p<0.0241). The equilibrium viscoelastic parameters show healthy discs encounter higher forces at the same strain level, and are able to maintain this force, where degenerated discs are unable to maintain this force throughout time. This morphology-conserved method provides insight into the spatial compressive mechanical properties of the intervertebral disc across the degeneration spectrum and will aid in modeling these tissue changes.     

A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells

Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.     

Perlecan, the multidomain heparan sulfate proteoglycan of basement membranes, is also a prominent component of the cartilaginous primordia in the developing human fetal spine.

The aim of this study was to localize perlecan in human fetal spine tissues. Human fetal spines (12-20 weeks; n=6) were fixed in either Histochoice or 10% neutral buffered formalin, routinely processed, paraffin-embedded, and 4-microm sagittal sections were cut and stained with toluidine blue, H&E, and von Kossa. Perlecan, types I, II, IV, and X collagen, CD-31, aggrecan core protein, and native and delta-HS 4, 5 hexuronate stub epitopes were immunolocalized. Toluidine blue staining visualized the cartilaginous vertebral body (VB) rudiments and annular lamellae encompassing the nucleus pulposus (NP). Von Kossa staining identified the VB primary center of ossification. Immunolocalization of type IV collagen, CD-31, and perlecan delineated small blood vessels in the outer annulus fibrosus (AF) and large canals deep within the VBs. Perlecan and type X collagen were also prominently expressed by the hypertrophic vertebral growth plate chondrocytes. Aggrecan was extracellularly distributed in the intervertebral disk (IVD) with intense staining in the posterior AF. Notochordal tissue stained strongly for aggrecan but negatively for perlecan and types I and II collagen. Type I collagen was prominent in the outer AF and less abundant in the NP, while type II collagen was localized throughout the IVD and VB. The immunolocalization patterns observed indicated key roles for perlecan in vasculogenic, chondrogenic, and endochondral ossification processes associated with spinal development.     

不同月龄大鼠椎间盘退变与多效生长因子表达的关系

目的观察不同月龄大鼠椎间盘的形态学变化并检测椎间盘中多效生长因子（pleiotrophin,PTN）的表达,探讨PTN与椎间盘退变的关系。方法取Wistar大鼠50只,以1,3,6,12,18个月龄不同分为5组,每组10只。采用苏木精-伊红染色观察椎间盘的形态学变化。采用SABC免疫组织化学方法,检测椎间盘中PTN的表达情况;结果（1）随着月龄的增加,椎间盘组织结构紊乱的程度逐渐增加,髓核内基质降解、正中出现空腔,胶原纤维增生、粗大、排列紊乱、并可见纤维断裂或缺失。（2）随着大鼠月龄的增加（1-12月龄）,椎间盘细胞中PTN的表达有逐渐减低的趋势,但至18月龄,PTN表达又有所增加;6和12月龄组椎间盘细胞中PTN的表达显著低于1月龄组,而18月龄组PTN的表达显著高于12月龄组。同月龄组椎间盘细胞中,PTN在终板的表达高于髓核和纤维环,髓核和纤维环中PTN的表达未见明显差异。结论大鼠椎间盘结构随月龄增加发生退行性变,PTN参与了大鼠椎间盘的退变,并可能通过促进椎间盘组织中新生血管的形成,延缓椎间盘的退变。     

Localization of cathepsins G and L in spontaneous resorption of intervertebral discs in a rat experimental model

To determine the involvement of cathepsins G and L in the mechanism of spontaneous resorption of herniated intervertebral discs, localization of these cathepsins in this process was examined immunohistochemically using a rat model of autologous transplantation of coccygeal discs. Rat coccygeal discs were resected and autotransplanted into the subcutaneous space of the skin of the back. Paraffin-embedded sections of intervertebral disc tissue, harvested at various post-transplantational periods, were immunohistochemically stained with antibodies for cathepsin G, cathepsin L, MMP-1, MMP-3 and ED-2. The number of positive cells was counted in each part of the transplanted discs. Immunolocalization of cathepsins G and L in various types of disc cells was first observed early in the post-transplantation period. From two days after the operation, histology showed invasion by granulation tissue, with many macrophages, in all sections. Subsequently, the number of macrophages in granulation tissue was observed to increase, along with a gradual increase in the percentage of cells positive for MMP-1 and MMP-3. In addition to the ability of cathepsins G and L to degrade major extracellular matrix components of intervertebral discs, cathepsin G is capable of activating latent pro-MMPs. The up-regulation of cathepsins G and L in the intervertebral disc tissue in this spontaneous resorption model suggests that these proteinases may be involved in degradation of extracellular matrix, leading to the natural resorption of herniated discs.     

Confocal microscopy demonstrates association of LTBP-2 in fibrillin-1 microfibrils and colocalisation with perlecan in the disc cell pericellular matrix

Comparative immunolocalisations of latent transforming growth factor-beta-1 binding protein (LTBP)-2, fibrillin-1, versican and perlecan were undertaken in foetal human and wild type C57BL/6 mouse and Hspg2 exon 3 null HS deficient mouse intervertebral discs (IVDs). LTBP-2 was a prominent pericellular component of annular fibrochondrocytes in the posterior annulus fibrosus (AF), interstitial matrix adjacent to nucleus pulposus (NP) cells and to fibrillar and cell associated material in the anterior AF of the human foetal IVD and also displayed a pericellular localisation pattern in murine IVDs. Perlecan and LTBP-2 displayed strong pericellular colocalisation patterns in the posterior AF and to fibrillar material in the outer anterior AF in the foetal human IVD. Versican was a prominent fibril-associated component in the posterior and anterior AF, localised in close proximity to fibrillin-1 in fibrillar arrangements in the cartilaginous vertebral rudiments around paraspinal blood vessels, to major collagen fibre bundles in the anterior and posterior AF and shorter fibres in the NP. Fibrillin-1 was prominent in the outer anterior AF of the human foetal IVD and in fibres extending from the AF into the cartilaginous vertebral rudiments. LTBP-2 was prominently associated with annular fibrils containing fibrillin-1, versican was localised in close proximity to these but not specifically with LTBP-2. The similar deposition levels of LTBP-2 observed in the AF of the Hspg2 exon 3 null and wild type murine IVDs indicated that perlecan HS was not essential for LTBP-2 deposition but colocalisation of LTBP-2 with perlecan in the foetal human IVD was consistent with HS mediated interactions which have already been demonstrated in-vitro.     

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes

Introduction  

Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.     

The composition and protein metabolism in the immature rabbit intervertebral disc

The nucleus pulposus (NP) and annulus fibrosus (AF) of immature rabbit intervertebral discs (IVD) have been subjected to the dissociative extraction procedure of Sajdera and Hascall (1969). The soluble, insoluble and unextracted fractions so obtained were analysed for total nitrogen, collagen, tyrosine, uronic acid, hexosamine and sialic acid content. A high proportion of non-collagenous protein, hexose and sialic acid in the NP insoluble fraction suggests the presence of glycopeptides associated with collagen and/or proteoglycans. The levels of proteoglycan in the soluble NP and AF fraction are similar. Immature (soluble) collagen, however, resides largely in the AF region. The metabolism of rabbit IVD protein components was also investigated both chemically and by autoradiography. L-Tyrosine-3,5-H3 was administered intraperitoneally (3 mc/kg) to 4 week-old rabbits. Animals were sacrificed at various time intervals and the harvested tissues extracted as before and lumbar discs collected. The levels of L-Tyrosine-3,5-H3 in the NP and AF insoluble and soluble fractions were determined using a tritium scintillation counting procedure and localisation by autoradiography. Pronounced extracellular activity of proteoglycan and glycoprotein is not evident before 24 hours. Soluble collagen, however, is synthesized and dispersed within 4 hours of isotope administration.     

Construction of tissue-engineered composite intervertebral disc and preliminary morphological and biochemical evaluation

The aim of this study was to construct tissue-engineered composite intervertebral disc (IVD) consisting of demineralized bone matrix gelatin (DBMG) and collagenII/hyaluronate/chondroitin-6-sulfate (CII/HyA–CS) scaffolds seeded with anulus fibrosus (AF) and nucleus pulposus (NP) cells, respectively. The cell-scaffold hybrids were implanted in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, the gross and histological morphology and biochemical properties were evaluated. Our results are as following: the gross morphology and histology of the composite resembled those of native IVD. Morphological studies revealed progressive tissue formation and junction integration between AF and NP regions. Biochemical composition detection indicated that the content of DNA, proteoglycan and hydroxyproline increased with time, and were similar to native tissue at 12 weeks. All these results demonstrated the feasibility of creating a tissue-engineered composite IVD with similar morphological and biochemical properties to the native tissue.     

缺氧诱导因子-1α和血管内皮生长因子在突出腰椎间盘中的表达及意义

目的探讨缺氧诱导因子-1α（hypoxia—induciblefactor-1α,HIF-1α）和血管内皮生长因子（vascular endothelial growth factor,VEGF）在突出腰椎间盘组织中的表达及意义。方法采用链霉亲和素-过氧化物酶复合物（SABC）免疫组化方法,测定40例腰椎间盘突出症患者椎间盘组织中HIF-1α和VEGF的表达情况。结果退变椎间盘组织中HIF-1α和VEGF呈高表达,HIF-1α和VEGF在髓核的表达显著高于纤维环;纤维环破裂型显著高于纤维环完整型;各组中HIF-1α和VEGF的表达均高度相关。结论HIF-1α和VEGF共同参与了椎间盘退变;HIF-1α可能通过上调VEGF的表达来促进椎间盘组织中新生血管的形成,进而延缓椎间盘退变的发生。