Novel ex-vivo mechanobiological intervertebral disc culture system

Intervertebral disc degeneration, a leading cause of low back pain, poses a significant socioeconomic burden with a broad array of costly treatment options. Mechanical loading is important in disease progression and treatment. Connecting mechanics and biology is critical for determining how loading parameters affect cellular response and matrix homeostasis. A novel ex-vivo experimental platform was developed to facilitate in-situ loading of rabbit functional spinal units (FSUs) with relevant biological outcome measures. The system was designed for motion outside of an incubator and validated for rigid fixation and physiologic environmental conditions. Specimen motion relative to novel fixtures was assessed using a digitizer; fixture stiffness exceeded specimen stiffness by an order of magnitude. Intradiscal pressure (IDP), measured using a fiber-optic pressure transducer, confirmed rigidity and compressive force selection. Surrounding media was controlled at 37 °C, 5% O(2)/CO(2) using a closed flow loop with an hypoxic incubator and was validated with probes in the specimen chamber. FSUs were subjected to cyclic compression (20 cycles) and four-hour creep at 1.0 MPa. Disc tissue was analyzed for cell viability using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which showed high viability (>90%) regardless of loading. Conditioned media was assayed for type-II collagen degradation fragments (CTX-II) and an aggrecan epitope (CS-846) associated with new aggrecan synthesis. CTX-II concentrations were not associated with loading, but CS-846 concentrations appeared to be increased with loading. Preservation of the full FSU allows physiologic load transmission and future multi-axis motion and identification of load-responsive proteins, thereby forming a new niche in intervertebral disc organ culture.     

Advances in susceptibility genetics of intervertebral degenerative disc disease

The traditional view that the etiology of lumbar disc herniation is primarily due to age, gender, occupation, smoking and exposure to vehicular vibration dominated much of the last century. Recent research indicates that heredity may be largely responsible for the degeneration as well as herniation of intervertebral discs. Since 1998, genetic influences have been confirmed by the identification of several genes forms associated with disc degeneration. These researches are paving the way for a better understanding of the biologic mechanisms. Now, many researchers unanimously agree that lumbar disc herniation appears to be similar to other complex diseases, whose etiology has both environmental and hereditary influence, each with a part of contribution and relative risk. Then addressing the etiological of lumbar disc herniation, it is important to integrate heredity with the environment factors. For the purpose of this review, we have limited our discussion to several susceptibility genes associated with disc degeneration.     

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.     

The effect of sustained compression on oxygen metabolic transport in the intervertebral disc decreases with degenerative changes

Intervertebral disc metabolic transport is essential to the functional spine and provides the cells with the nutrients necessary to tissue maintenance. Disc degenerative changes alter the tissue mechanics, but interactions between mechanical loading and disc transport are still an open issue. A poromechanical finite element model of the human disc was coupled with oxygen and lactate transport models. Deformations and fluid flow were linked to transport predictions by including strain-dependent diffusion and advection. The two solute transport models were also coupled to account for cell metabolism. With this approach, the relevance of metabolic and mechano-transport couplings were assessed in the healthy disc under loading-recovery daily compression. Disc height, cell density and material degenerative changes were parametrically simulated to study their influence on the calculated solute concentrations. The effects of load frequency and amplitude were also studied in the healthy disc by considering short periods of cyclic compression. Results indicate that external loads influence the oxygen and lactate regional distributions within the disc when large volume changes modify diffusion distances and diffusivities, especially when healthy disc properties are simulated. Advection was negligible under both sustained and cyclic compression. Simulating degeneration, mechanical changes inhibited the mechanical effect on transport while disc height, fluid content, nucleus pressure and overall cell density reductions affected significantly transport predictions. For the healthy disc, nutrient concentration patterns depended mostly on the time of sustained compression and recovery. The relevant effect of cell density on the metabolic transport indicates the disturbance of cell number as a possible onset for disc degeneration via alteration of the metabolic balance. Results also suggest that healthy disc properties have a positive effect of loading on metabolic transport. Such relation, relevant to the maintenance of the tissue functional composition, would therefore link disc function with disc nutrition.     

Persistent degenerative changes in thymic organ function revealed by an inducible model of organ regrowth

The thymus is the most rapidly aging tissue in the body, with progressive atrophy beginning as early as birth and not later than adolescence. Latent regenerative potential exists in the atrophic thymus, because certain stimuli can induce quantitative regrowth, but qualitative function of T lymphocytes produced by the regenerated organ has not been fully assessed. Using a genome-wide computational approach, we show that accelerated thymic aging is primarily a function of stromal cells, and that while overall cellularity of the thymus can be restored, many other aspects of thymic function cannot. Medullary islet complexity and tissue-restricted antigen expression decrease with age, representing potential mechanisms for age-related increases in autoimmune disease, but neither of these is restored by induced regrowth, suggesting that new T cells produced by the regrown thymus will probably include more autoreactive cells. Global analysis of stromal gene expression profiles implicates widespread changes in Wnt signaling as the most significant hallmark of degeneration, changes that once again persist even at peak regrowth. Consistent with the permanent nature of age-related molecular changes in stromal cells, induced thymic regrowth is not durable, with the regrown organ returning to an atrophic state within 2 weeks of reaching peak size. Our findings indicate that while quantitative regrowth of the thymus is achievable, the changes associated with aging persist, including potential negative implications for autoimmunity.     

An extended biphasic model for charged hydrated tissues with application to the intervertebral disc

Finite element models for hydrated soft biological tissue are numerous but often exhibit certain essential deficiencies concerning the reproduction of relevant mechanical and electro-chemical responses. As a matter of fact, singlephasic models can never predict the interstitial fluid flow or related effects like osmosis. Quite a few models have more than one constituent, but are often restricted to the small-strain domain, are not capable of capturing the intrinsic viscoelasticity of the solid skeleton, or do not account for a collagen fibre reinforcement. It is the goal of this contribution to overcome these drawbacks and to present a thermodynamically consistent model, which is formulated in a very general way in order to reproduce the behaviour of almost any charged hydrated tissue. Herein, the Theory of Porous Media (TPM) is applied in combination with polyconvex Ogden-type material laws describing the anisotropic and intrinsically viscoelastic behaviour of the solid matrix on the basis of a generalised Maxwell model. Moreover, other features like the deformation-dependent permeability, the possibility to include inhomogeneities like varying fibre alignment and behaviour, or osmotic effects based on the simplifying assumption of Lanir are also included. Finally, the human intervertebral disc is chosen as a representative for complex soft biological tissue behaviour. In this regard, two numerical examples will be presented with focus on the viscoelastic and osmotic capacity of the model.     

An ionised/non-ionised dual porosity model of intervertebral disc tissue

The volume of the intrafibrillar water space – i.e. the water contained inside the collagen fibres – is a key parameter that is relevant to concepts of connective tissue structure and function. Confined compression and swelling experiments on annulus fibrosus samples are interpreted in terms of a dual porosity model that distinguishes between a non-ionised intrafibrillar porosity and an ionised extrafibrillar porosity. Both porosities intercommunicate and are saturated with a monovalent ionic solution, i.c. NaCl. The extrafibrillar fixed charge density of the samples is assessed using radiotracer techniques and the collagen content is evaluated by measurement of hydroxyproline concentration. The interpretation of the experimental data yields values for the intrafibrillar water content, the average activity coefficient of the ions, the Donnan osmotic coefficient, the fraction of intrafibrillar water, the stress-free deformation state, and an effective stress–strain relationship as a function of the radial position in the disc. A linear fit between the second Piola–Kirchhoff effective stress and Green–Lagrange strain yielded an effective stiffness: H_e=1.087 ± 0.657 MPa. The average fraction of intrafibrillar water was 1.16 g/g collagen. The results were sensitive to changes in the activity and osmotic coefficients and the fraction of intrafibrillar water. The fixed charge density increased with distance from the outer edge of the annulus, whereas the hydroxyproline decreased.The authors wish to thank Dr. Jill Urban for her advice concerning fixed charge density measurements, and Ing. Paul Willems for his assistance with the experiments. The research of Dr. J. M. Huyghe has been made possible through a fellowship of the Royal Netherlands Academy of Arts and Sciences.     

The biological basis of degenerative disc disease: proteomic and biomechanical analysis of the canine intervertebral disc

IntroductionIn the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP).MethodsWe used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines.ResultsWe detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion.ConclusionsThe CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0733-z) contains supplementary material, which is available to authorized users.     

The effect of degenerative morphological changes of the intervertebral disc on the lumbar spine biomechanics: a poroelastic finite element investigation

Intervertebral disc degeneration involves changes in the spinal anatomical structures. The mechanical relevance of the following changes was investigated: disc height, endplate sclerosis, disc water content, permeability and depressurisation. A poroelastic nonlinear finite element model of the L4–L5 human spine segments was employed. Loads represented a daily cycle (500 N compression combined with flexion–extension motion for 16 h followed by 200 N compression for 8 h). In non-degenerative conditions, the model predicted a diurnal axial displacement of 1.32 mm and a peak intradiscal pressure of 0.47 MPa. Axial displacement, facet force and range of motion in flexion–extension are decreased by decreasing disc height. By decreasing the initial water content, axial displacement, facet force and fluid loss were all reduced. Endplate sclerosis did not have a significant influence on the calculated results. Depressurisation determined an increase of the disc effective stress, possibly inducing failure. Degenerative instability was not calculated in any simulations.     

A mechanical model for the human intervertebral disc

Stress relaxation experiments were performed on specimens from a human intervertebral disc. Specimens were made from the nucleus pulposus and from the external lamellae of the anulus fibrosus in two different orientations. Tests were run with varying moisture content so as to develop a relaxation master curve. A model was developed based on the experimental data. It was found that the short term master curve for the lamellae of the anulus and nucleus are similar, whereas the long term rubbery plateau is different between the lamellae and the nucleus. It was also established that the master curves for different lamellae and the nucleus were shifted relative to each other in the time domain due to changes in water content. The average relaxation modulus of the whole disc was obtained by averaging the properties between the anulus and nucleus. This model was then used for studies of Schmorl's nodes, of degenerated discs and for circumstances in which hydration is considered to be important.     

The effect of degenerative morphological changes of the intervertebral disc on the lumbar spine biomechanics: a poroelastic finite element investigation

Intervertebral disc degeneration involves changes in the spinal anatomical structures. The mechanical relevance of the following changes was investigated: disc height, endplate sclerosis, disc water content, permeability and depressurisation. A poroelastic nonlinear finite element model of the L4-L5 human spine segments was employed. Loads represented a daily cycle (500 N compression combined with flexion-extension motion for 16 h followed by 200 N compression for 8 h). In non-degenerative conditions, the model predicted a diurnal axial displacement of 1.32 mm and a peak intradiscal pressure of 0.47 MPa. Axial displacement, facet force and range of motion in flexion-extension are decreased by decreasing disc height. By decreasing the initial water content, axial displacement, facet force and fluid loss were all reduced. Endplate sclerosis did not have a significant influence on the calculated results. Depressurisation determined an increase of the disc effective stress, possibly inducing failure. Degenerative instability was not calculated in any simulations.     

Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes

The expression of the matrix protein chondromodulin-I has been studied in human intervertebral discs of 101 people using immunohistochemical analyses. The purpose of this report is to present data on the metabolic changes that were found to occur in the chondrocytes of intervertebral discs during development and aging. Chondromodulin-I was highly expressed during the gestational period and gradually decreased after maturation. It was detected in both the extracellular matrix and chondrocytes in the zone of hypertrophic cartilage, the zone of proliferative cartilage and the zone of resting cartilage in fetal discs. It was also present in the annulus fibrosus, nucleus pulposus and end-plate cartilage in mature discs. In degenerative discs, chondromodulin-I immunoreactivity tended to be elevated in the remaining chondrocytes. Our findings suggest that the expression of the protein is developmentally regulated and upregulated through a defense mechanism against the degenerative processes of the aged intervertebral disc.     

An organ culture system for study of fetal lung development

Fetal rat lungs placed in $\text{in}$$\text{vitro}$ organ culture at 15.5 days gestation grow significantly based on accumulation of DNA and protein. In the experimental system described, DNA accumulated rapidly during the first three days in culture and increased from 4.8 to 15.6 micrograms per lung culture. Protein content increased more slowly and reached a value more than double the initial value after six days in the culture system. Glycogen accumulated in the tissue during the first six days in culture and was depleted during the subsequent culture period, a pattern strikingly similar to that observed during lung development $\text{in}$$\text{vivo}$. Phospholipid accumulation was biphasic with respect to time with an inflection point at about the sixth day of culture. The phosphatidylcholine species synthesized in the culture system $\text{in}$$\text{vitro}$ were similar to those produced $\text{in}$$\text{vivo}$ in fetal lung at 21 days gestation.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

Degeneration of the intervertebral disc

The intervertebral disc is a cartilaginous structure that resembles articular cartilage in its biochemistry, but morphologically it is clearly different. It shows degenerative and ageing changes earlier than does any other connective tissue in the body. It is believed to be important clinically because there is an association of disc degeneration with back pain. Current treatments are predominantly conservative or, less commonly, surgical; in many cases there is no clear diagnosis and therapy is considered inadequate. New developments, such as genetic and biological approaches, may allow better diagnosis and treatments in the future.     

Biological response of the intervertebral disc to dynamic loading

Disc degeneration is a chronic remodeling process that results in alterations of matrix composition and decreased cellularity. This study tested the hypothesis that dynamic mechanical forces are important regulators in vivo of disc cellularity and matrix synthesis. A murine model of dynamic loading was developed that used an external loading device to cyclically compress a single disc in the tail. Loads alternated at a 50% duty cycle between 0MPa and one of two peak stresses (0.9 or 1.3MPa) at one of two frequencies (0.1 or 0.01Hz) for 6h per day for 7 days. An additional group received static compression at 1.3MPa for 3h/day for 7 days. A control group wore the device with no loading. Sections of treated discs were analyzed for morphology, proteoglycan content, apoptosis, cell areal density, and aggrecan and collagen II gene expression. Dynamic loading induced differential effects that depended on frequency and stress. No significant changes to morphology, proteoglycan content or cell death were found after loading at 0.9MPa, 0.1Hz. Loading at lower frequency and/or higher stress increased proteoglycan content, matrix gene expression and cell death. The results have implications in the prevention of intervertebral disc degeneration, suggesting that loading conditions may be optimized to promote maintenance of normal structure and function.     

Pathophysiology of the human intervertebral disc

Intervertebral disc degeneration is a common invalidating disorder that can affect the musculoskeletal apparatus in both younger and older ages. The chief component of the intervertebral disc is the highly organized extracellular matrix; maintenance of its organization is essential for correct spinal mechanics. The matrix components, mainly proteoglycans and collagens, undergo a slow and continuous cell-mediated turnover process that enables disc cells to adapt their environment to external stimuli. Cellular senescence and a history of chronic abnormal loading can upset this balance, leading to progressive tissue failure that results in disc degeneration. Although biological treatment approaches to disc repair are still far to come, advances in our understanding of disc biochemistry and in defining the role of genetic inheritance have provided a starting point for developing new concepts in the diagnosis, therapy and prevention of disc degeneration.