Role of load history in intervertebral disc mechanics and intradiscal pressure generation

Solid–fluid interactions play an important role in mediating viscoelastic behaviour of biological tissues. In the intervertebral disc, water content is governed by a number of factors, including age, disease and mechanical loads, leading to changes in stiffness characteristics. We hypothesized that zonal stress distributions depend on load history, or the prior stresses experienced by the disc. To investigate these effects, rat caudal motion segments were subjected to compressive creep biomechanical testing in vitro using a protocol that consisted of two phases: a Prestress Phase (varied to represent different histories of load) followed immediately by an Exertion Phase, identical across all Prestress groups. Three analytical models were used to fit the experimental data in order to evaluate load history effects on gross and zonal disc mechanics. Model results indicated that while gross transient response was insensitive to load history, there may be changes in the internal mechanics of the disc. In particular, a fluid transport model suggested that the role of the nucleus pulposus in resisting creep during Exertion depended on Prestress conditions. Separate experiments using similarly defined load history regimens were performed to verify these predictions by measuring intradiscal pressure with a fibre optic sensor. We found that the ability for intradiscal pressure generation was load history-dependent and exhibited even greater sensitivity than predicted by analytical models. A 0.5?MPa Exertion load resulted in 537.2?kPa IDP for low magnitude Prestress compared with 373.7?kPa for high magnitude Prestress. Based on these measurements, we developed a simple model that may describe the pressure-shear environment in the nucleus pulposus. These findings may have important implications on our understanding of how mechanical stress contributes to disc health and disease etiology.     

The Effect of Nucleus Pulposus Crosslinking and Glycosaminoglycan Degradation on Disc Mechanical Function

Altered mechanical loading, secondary to biochemical changes in the nucleus pulposus, is a potential mechanism in disc degeneration. An understanding of the role of this altered mechanical loading is only possible by separating the mechanical and biological effects of early nucleus pulposus changes. The objective of this study was to quantify the mechanical effect of decreased glycosaminoglycans (GAG) and increased crosslinking in the nucleus pulposus using in vitro rat lumbar discs. Following initial mechanical testing the discs were injected according to the four treatment groups: PBS control, chondroitinase-ABC (ChABC) for GAG degradation, genipin (Gen) for crosslinking, or a combination of chondroitinase and genipin (ChABC+Gen). After treatment the discs were again mechanically tested, followed by histology or biochemistry. Neutral zone mechanical properties were changed by approximately 20% for PBS, ChABC, and ChABC+Gen treatments (significant only for PBS in a paired comparison). These trends were reversed with genipin crosslinking alone. With ChABC treatment the effective compressive modulus increased and the GAG content decreased; with the combination of ChABC+Gen the mechanics and GAG content were unchanged. Degradation of nucleus pulposus GAG alters disc axial mechanics, potentially contributing to the degenerative cascade. Crosslinking is unlikely to contribute to degeneration, but may be a potential avenue of treatment.     

A Structurally and Functionally Biomimetic Biphasic Scaffold for Intervertebral Disc Tissue Engineering

Tissue engineering offers high hopes for the treatment of intervertebral disc (IVD) degeneration. Whereas scaffolds of the disc nucleus and annulus have been extensively studied, a truly biomimetic and mechanically functional biphasic scaffold using naturally occurring extracellular matrix is yet to be developed. Here, a biphasic scaffold was fabricated with collagen and glycosaminoglycans (GAGs), two of the most abundant extracellular matrix components in the IVD. Following fabrication, the scaffold was characterized and benchmarked against native disc. The biphasic scaffold was composed of a collagen-GAG co-precipitate making up the nucleus pulposus-like core, and this was encapsulated in multiple lamellae of photochemically crosslinked collagen membranes comprising the annulus fibrosus-like lamellae. On mechanical testing, the height of our engineered disc recovered by ~82-89% in an annulus-independent manner, when compared with the 99% recovery exhibited by native disc. The annulus-independent nature of disc height recovery suggests that the fluid replacement function of the engineered nucleus pulposus core might mimic this hitherto unique feature of native disc. Biphasic scaffolds comprised of 10 annulus fibrosus-like lamellae had the best overall mechanical performance among the various designs owing to their similarity to native disc in most aspects, including elastic compliance during creep and recovery, and viscous compliance during recovery. However, the dynamic mechanical performance (including dynamic stiffness and damping factor) of all the biphasic scaffolds was similar to that of the native discs. This study contributes to the rationalized design and development of a biomimetic and mechanically viable biphasic scaffold for IVD tissue engineering.     

Penetrating annulus fibrosus injuries affect dynamic compressive behaviors of the intervertebral disc via altered fluid flow: an analytical interpretation

Extensive experimental work on the effects of penetrating annular injuries indicated that large injuries impact axial compressive properties of small animal intervertebral discs, yet there is some disagreement regarding the sensitivity of mechanical tests to small injury sizes. In order to understand the mechanism of injury size sensitivity, this study proposed a simple one dimensional model coupling elastic deformations in the annulus with fluid flow into and out of the nucleus through both porous boundaries and through a penetrating annular injury. The model was evaluated numerically in dynamic compression with parameters obtained by fitting the solution to experimental stress-relaxation data. The model predicted low sensitivity of mechanical changes to injury diameter at both small and large sizes (as measured by low and high ratios of injury diameter to annulus thickness), with a narrow range of high sensitivity in between. The size at which axial mechanics were most sensitive to injury size (i.e., critical injury size) increased with loading frequency. This study provides a quantitative hypothetical model of how penetrating annulus fibrosus injuries in discs with a gelatinous nucleus pulposus may alter disc mechanics by changing nucleus pulposus fluid pressurization through introduction of a new fluid transport pathway though the annulus. This model also explains how puncture-induced biomechanical changes depend on both injury size and test protocol.     

The influence of exogenous cross-linking and compressive creep loading on intradiscal pressure

This study involves a biomechanical evaluation of a prospective injectable treatment for degenerative discs. The high osmolarity of the non-degenerated nucleus pulposus attracts water contributing to the hydrostatic behavior of the tissue. This intradiscal pressure is known to drop as fluid is exuded from the matrix due to compressive loading. The objective of this study was to compare the changes in intradiscal pressure in control and genipin cross-linked intervertebral discs. Thirty bovine lumbar motion segments were randomly divided into a phosphate-buffered saline control group and a 0.33% genipin group and soaked at room temperature for 2 days. A needle pressure sensor was held in the center of the disc while short-term and static creep compressive loads were applied. The control group demonstrated a 25% higher average intradiscal pressure compared to genipin-treated discs under 750 N compressive load (p=0.029). Depressurization during static compressive creep was 56% higher in the control than in the genipin group (p=0.014). These results suggest cross-linking induced changes in the poroelastic properties of the involved tissues affected the mechanics of compressive load support in the disc with lower levels of nucleus pressure, a corresponding decrease in the elastic expansion of the annulus, and an increased axial compressive loading of the inner and outer annulus tissues. It is possible that concurrent changes in hydraulic permeability and proteoglycan retention known to be associated with genipin cross-linking were also contributors to poroelastic changes. Reduction of peak pressures and moderation of pressure fluctuations could be beneficial relative to discogenic pain.     

Nonlinear finite element analysis of anular lesions in the L4/5 intervertebral disc

Degenerate intervertebral discs exhibit both material and structural changes. Structural defects (lesions) develop in the anulus fibrosus with age. While degeneration has been simulated in numerous previous studies, the effects of structural lesions on disc mechanics are not well known. In this study, a finite element model (FEM) of the L4/5 intervertebral disc was developed in order to study the effects of anular lesions and loss of hydrostatic pressure in the nucleus pulposus on the disc mechanics. Models were developed to simulate both healthy and degenerate discs. Degeneration was simulated with either rim, radial or circumferential anular lesions and by equating nucleus pressure to zero. The anulus fibrosus ground substance was represented as a nonlinear incompressible material using a second-order polynomial, hyperelastic strain energy equation. Hyperelastic material parameters were derived from experimentation on sheep discs. Endplates were assumed to be rigid, and annulus lamellae were assumed to be vertical in the unloaded state. Loading conditions corresponding to physiological ranges of rotational motion were applied to the models and peak rotation moments compared between models. Loss of nucleus pulposus pressure had a much greater effect on the disc mechanics than the presence of anular lesions. This indicated that the development of anular lesions alone (prior to degeneration of the nucleus) has minimal effect on disc mechanics, but that disc stiffness is significantly reduced by the loss of hydrostatic pressure in the nucleus. With the degeneration of the nucleus, the outer innervated anulus or surrounding osteo-ligamentous anatomy may therefore experience increased strains.     

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.     

Swelling pressure of the inervertebral disc: influence of proteoglycan and collagen contents

In this study we have considered how equilibrium water content of the human nucleus pulposus varies with applied pressure for discs of various spinal levels and of various ages. In all cases hydration decreased as pressure increased but the level of equilibrium hydration depended on the relative amounts of collagen and PG in the tissue. Provided we accounted for the exclusion of PGs from the intra-fibrillar space, the swelling pressure curve and the osmotic pressure curve of equivalent PGs were found to coincide. The result implies that under physiological hydrations the mechanical forces exerted by the collagen network of the nucleus are insignificant and that the osmotic pressure of the proteoglycans is balanced by the applied pressure arising from body weight and muscle and ligament tension alone. Since aged discs often have a low proteoglycan to collagen ratio, their equilibrium hydration also tends to be low. Moreover a far larger proportion of the total water is associated with the collagen than in the younger disc.     

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     

Periostin is expressed by cells of the human and sand rat intervertebral discs

Abstract

Periostin, a matricellular protein in the fasciclin family, is expressed in tissues subjected to constant mechanical stress. Periostin modulates cell-to-extracellular matrix interactions and can bind to collagen, fibronectin, tenascin-C and several integrins. Our objective was to evaluate whether periostin is expressed in the human intervertebral disc. Immunohistochemical localization of periostin was carried out in tissue of human lumbar discs and lumbar discs of the sand rat (Psammomys obesus). Human discs also were examined for periostin gene expression. Immunohistochemical localization demonstrated periostin in the cytoplasm of annulus and nucleus cells, and occasionally in the surrounding pericellular and interterritorial extracellular matrix. Periostin distribution in the human disc was distinctive. Outer annulus contained the highest proportion of periostin-positive cells (88.8%), whereas inner annulus contained only 61.4%. The nucleus pulposus contained the fewest periostin-positive cells (18.5%). There was a significant negative correlation between the percentage of cells positive for periostin in the inner annulus and subject age. Periostin gene expression in the human disc also was confirmed using molecular microarray analysis. Because work by others has shown that periostin plays an important role in the biomechanical properties of other connective tissues (skin, tendon, heart valves), future research is needed to elucidate the role of periostin in disc, loading, aging and degeneration.     

Effect of microgravity on the biomechanical properties of lumbar and caudal intervertebral discs in mice

Prolonged exposure to microgravity has shown to have deleterious effects on the human spine, indicated by low back pain during spaceflight and increased incidence of post-spaceflight herniated nucleus pulposus. We examined the effect of microgravity on biomechanical properties of lumbar and caudal discs from mice having been on 15-day shuttle mission STS-131. Sixteen C57BL/C mice (spaceflight group, n=8; ground-based control group, n=8) were sacrificed immediately after spaceflight. Physiological disc height (PDH) was measured in situ, and compressive creep tests were performed to parameterize biomechanical properties into endplate permeability (k), nuclear swelling pressure strain dependence (D), and annular viscoelasticity (G). For caudal discs, the spaceflight group exhibited 32% lower PDH, 70% lower D and crept more compared to the control mice (p=0.03). For lumbar discs, neither PDH nor D was significantly different between murine groups. Initial modulus, osmotic pressure, k and G for lumbar and caudal discs did not appear influenced by microgravity (p>0.05). Decreases in both PDH and D suggest prolonged microgravity effectively diminished biomechanical properties of caudal discs. By contrast, differences were not noted for lumbar discs. This potentially deleterious interaction between prolonged weightlessness and differential ranges of motion along the spine may underlie the increased cervical versus lumbar disc herniation rates observed among astronauts.     

Nucleotomy reduces the effects of cyclic compressive loading with unloaded recovery on human intervertebral discs

The first objective of this study was to determine the effects of physiological cyclic loading followed by unloaded recovery on the mechanical response of human intervertebral discs. The second objective was to examine how nucleotomy alters the disc?s mechanical response to cyclic loading. To complete these objectives, 15 human L5-S1 discs were tested while intact and subsequent to nucleotomy. The testing consisted of 10,000 cycles of physiological compressive loads followed by unloaded hydrated recovery. Cyclic loading increased compression modulus (3%) and strain (33%), decreased neutral zone modulus (52%), and increased neutral zone strain (31%). Degeneration was not correlated with the effect of cyclic loading in intact discs, but was correlated with cyclic loading effects after nucleotomy, with more degenerate samples experiencing greater increases in both compressive and neutral zone strain following cyclic loading. Partial removal of the nucleus pulposus decreased the compression and neutral zone modulus while increasing strain. These changes correspond to hypermobility, which will alter overall spinal mechanics and may impact low back pain via altered motion throughout the spinal column. Nucleotomy also reduced the effects of cyclic loading on mechanical properties, likely due to altered fluid flow, which may impact cellular mechanotransduction and transport of disc nutrients and waste. Degeneration was not correlated with the acute changes of nucleotomy. Results of this study provide an ideal protocol and control data for evaluating the effectiveness of a mechanically-based disc degeneration treatment, such as a nucleus replacement.     

Disc mechanics with trans-endplate partial nucleotomy are not fully restored following cyclic compressive loading and unloaded recovery

Mechanical function of the intervertebral disc is maintained through the interaction between the hydrated nucleus pulposus, the surrounding annulus fibrosus, and the superior and inferior endplates. In disc degeneration the normal transfer of load between disc substructures is compromised. The objective of this study was to explore the mechanical role of the nucleus pulposus in support of axial compressive loads over time. This was achieved by measuring the elastic slow ramp and viscoelastic stress-relaxation mechanical behaviors of cadaveric sheep motion segments before and after partial nucleotomy through the endplate (keeping the annulus fibrosus intact). Mechanics were evaluated at five conditions: Intact, intact after 10,000 cycles of compression, acutely after nucleotomy, following nucleotomy and 10,000 cycles of compression, and following unloaded recovery. Radiographs and magnetic resonance images were obtained to examine structure. Only the short time constant of the stress relaxation was altered due to nucleotomy. In contrast, cyclic loading resulted in significant and large changes to both the stiffness and stress relaxation behaviors. Moreover, the nucleotomy had little to no effect on the disc mechanics after cyclic loading, as there were no significant differences comparing mechanics after cyclic loading with or without the nucleotomy. Following unloaded recovery the mechanical changes that had occurred as a consequence of cyclic loading were restored, leaving only a sustained change in the short time constant due to the trans-endplate nucleotomy. Thus the swelling and redistribution of the remaining nucleus pulposus was not able to fully restore mechanical behaviors. This study reveals insights into the role of the nucleus pulposus in disc function, and provides new information toward the potential role of altered nucleus pulpous function in the degenerative cascade.     

Human disc degeneration is associated with increased MMP 7 expression.

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Human disc degeneration is associated with increased MMP 7 expression

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Human disc degeneration is associated with increased MMP 7 expression

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Chondrocyte‐like nested cells in the aged intervertebral disc are late‐stage nucleus pulposus cells

Aging is a major risk factor of intervertebral disc degeneration and a leading cause of back pain. Pathological changes associated with disc degeneration include the absence of large, vacuolated and reticular‐shaped nucleus pulposus cells, and appearance of smaller cells nested in lacunae. These small nested cells are conventionally described as chondrocyte‐like cells; however, their origin in the intervertebral disc is unknown. Here, using a genetic mouse model and a fate mapping strategy, we have found that the chondrocyte‐like cells in degenerating intervertebral discs are, in fact, nucleus pulposus cells. With aging, the nucleus pulposus cells fuse their cell membranes to form the nested lacunae. Next, we characterized the expression of sonic hedgehog (SHH), crucial for the maintenance of nucleus pulposus cells, and found that as intervertebral discs age and degenerate, expression of SHH and its target Brachyury is gradually lost. The results indicate that the chondrocyte‐like phenotype represents a terminal stage of differentiation preceding loss of nucleus pulposus cells and disc collapse.     

The role of viscoelasticity of collagen fibers in articular cartilage: theory and numerical formulation

The relative importance of fluid-dependent and fluid-independent transient mechanical behavior in articular cartilage was examined for tensile and unconfined compression testing using a fibril reinforced model. The collagen matrix of articular cartilage was modeled as viscoelastic using a quasi-linear viscoelastic formulation with strain-dependent elastic modulus, while the proteoglycan matrix was considered as linearly elastic. The collagen viscoelastic properties were obtained by fitting experimental data from a tensile test. These properties were used to investigate unconfined compression testing, and the sensitivity of the properties was also explored. It was predicted that the stress relaxation observed in tensile tests was not caused by fluid pressurization at the macroscopic level. A multi-step tensile stress relaxation test could be approximated using a hereditary integral in which the elastic fibrillar modulus was taken to be a linear function of the fibrillar strain. Applying the same formulation to the radial fibers in unconfined compression, stress relaxation could not be simulated if fluid pressurization were absent. Collagen viscoelasticity was found to slightly weaken fluid pressurization in unconfined compression, and this effect was relatively more significant at moderate strain rates. Therefore, collagen viscoelasticity appears to play an import role in articular cartilage in tensile testing, while fluid pressurization dominates the transient mechanical behavior in compression. Collagen viscoelasticity plays a minor role in the mechanical response of cartilage in unconfined compression if significant fluid flow is present.