Matrix modification for enhancing the transport properties of the human cartilage endplate to improve disc nutrition.

Poor solute transport through the cartilage endplate (CEP) impairs disc nutrition and could be a key factor that limits the success of intradiscal biologic therapies. Here we demonstrate that treating the CEP with matrix metalloproteinase-8 (MMP-8) reduces the matrix constituents that impede solute uptake and thereby improves nutrient diffusion. Human CEP tissues harvested from four fresh cadaveric lumbar spines (age range: 38-66 years old) were treated with MMP-8. Treatment caused a dose-dependent reduction in sGAG, localized reductions to the amount of collagen, and alterations to collagen structure. These matrix modifications corresponded with 16-24% increases in the uptake of a small solute (376 Da). Interestingly, the effects of MMP-8 treatment depended on the extent of non-enzymatic glycation: treated CEPs with high concentrations of advanced glycation end products (AGEs) exhibited the lowest uptake compared to treated CEPs with low concentrations of AGEs. Moreover, AGE concentrations were donor-specific, and the donor tissues with the highest AGE concentrations appeared to have lower uptake than would be expected based on the initial amounts of collagen and sGAG. Finally, increasing solute uptake in the CEP improved cell viability inside diffusion chambers, which supports the nutritional relevance of enhancing the transport properties of the CEP. Taken together, our results provide new insights and in vitro proof-of-concept for a treatment approach that could improve disc nutrition for biologic therapy: specifically, matrix reduction by MMP-8 can enhance solute uptake and nutrient diffusion through the CEP, and AGE concentration appears to be an important, patient-specific factor that influences the efficacy of this approach

Mohawk promotes the maintenance and regeneration of the outer annulus fibrosus of intervertebral discs.

The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus fibrosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx(-/-) mice, the OAF displays a deficiency of multiple tendon/ligament-related genes, a smaller OAF collagen fibril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen fibril formation. Our results indicate a therapeutic strategy for AF regeneration

Comparison of Apparent Diffusion Coefficient and T2 Relaxation Time Variation Patterns in Assessment of Age and Disc Level Related Intervertebral Disc Changes

Purpose To compare the variation patterns of ADC and T2 values in different age and intervertebral disc (IVD) levels, thus to identify their sensitivities in assessing age and disc level related IVDs changes. Materials and Methods The T2 and ADC values were recorded from 345 IVDs of 69 volunteers. Kendall's correlation analysis was used to identify the relationship between age and T2/ADC mean values respectively. The one-way analysis of variance (ANOVA) with post hoc analysis was then applied to test the differences of T2 and ADC values among different IVD levels and age groups, followed by linear regression analysis between age (45 years) and T2/ADC mean values. This study was approved by the Ethics Committee of the Chinese Academy of Medical Sciences and the Peking Union Medical College Hospital. Results: Significant negative correlation was observed between age and T2/ADC mean values. The T2 and ADC values showed significant differences among IVD levels and among age groups except for T2 values in age group 1 (25–34 years) and group 2 (35–44 years), and for ADC values at L1–2 level. Both T2 and ADC values showed significant differences between young (age45 years) at each IVD level. A linear relationship was observed between age and T2/ADC mean values in the elderly group as well as in the young group for the ADC mean values, while no such tendency was identified in the young group for the T2 mean values. Conclusions: ADC values may be a more sensitive parameter than T2 in assessing age and disc level related intervertebral disc changes

Early changes in the extracellular matrix of the degenerating intervertebral disc, assessed by Fourier transform infrared imaging.

Mechanical overloading induces a degenerative cell response in the intervertebral disc. However, early changes in the extracellular matrix (ECM) are challenging to assess with conventional techniques. Fourier Transform Infrared (FTIR) imaging allows visualization and quantification of the ECM. We aim to identify markers for disc degeneration and apply these to investigate early degenerative changes due to overloading and katabolic cell activity. Three experiments were conducted; Exp 1.: In vivo, lumbar spines of seven goats were operated: one disc was injected with chondroitinase ABC (mild degeneration) and compared to the adjacent disc (control) after 24 weeks. Exp 2a: Ex vivo, caprine discs received physiological loading (n=10) or overloading (n=10) in a bioreactor. Exp 2b: Cell activity was diminished prior to testing by freeze-thaw cycles, 18 discs were then tested as in Exp 2a. In all experiments, FTIR images (spectral region: 1000-1300 cm ) of mid-sagittal slices were analyzed using multivariate curve resolution. In vivo, FTIR was more sensitive than biochemical and histological analysis in identifying reduced proteoglycan content (p=0.046) and increased collagen content in degenerated discs (p<0.01). Notably, FTIR analysis additionally showed disorganization of the ECM, indicated by increased collagen entropy (p=0.011). Ex vivo, the proteoglycan/collagen ratio decreased due to overloading (p=0.047) and collagen entropy increased (p=0.047). Cell activity affected collagen content only (p=0.044). FTIR imaging allows a more detailed investigation of early disc degeneration than traditional measures. Changes due to mild overloading could be assessed and quantified. Matrix remodeling is the first detectable step towards intervertebral disc degeneration. [Abstract copyright: Copyright © 2018. Published by Elsevier Ltd.

Histological analysis of surgical lumbar intervertebral disc tissue provides evidence for an association between disc degeneration and increased body mass index

<p>Abstract</p> <p>Background</p> <p>Although histopathological grading systems for disc degeneration are frequently used in research, they are not yet integrated into daily care routine pathology of surgical samples. Therefore, data on histopathological changes in surgically excised disc material and their correlation to clinical parameters such as age, gender or body mass index (BMI) is limited to date. The current study was designed to correlate major physico-clinical parameters from a population of orthopaedic spine center patients (gender, age and BMI) with a quantitative histologic degeneration score (HDS).</p> <p>Methods</p> <p>Excised lumbar disc material from 854 patients (529 men/325 women/mean age 56 (15-96) yrs.) was graded based on a previously validated histologic degeneration score (HDS) in a cohort of surgical disc samples that had been obtained for the treatment of either disc herniation or discogenic back pain. Cases with obvious inflammation, tumor formation or congenital disc pathology were excluded. The degree of histological changes was correlated with sex, age and BMI.</p> <p>Results</p> <p>The HDS (0-15 points) showed significantly higher values in the nucleus pulposus (NP) than in the annulus fibrosus (AF) (Mean: NP 11.45/AF 7.87), with a significantly higher frequency of histomorphological alterations in men in comparison to women. Furthermore, the HDS revealed a positive significant correlation between the BMI and the extent of histological changes. No statistical age relation of the degenerative lesions was seen.</p> <p>Conclusions</p> <p>This study demonstrated that histological disc alterations in surgical specimens can be graded in a reliable manner based on a quantitative histologic degeneration score (HDS). Increased BMI was identified as a positive risk factor for the development of symptomatic, clinically significant disc degeneration.</p

Degeneration of the intervertebral disc with new approaches for treating low back pain.

This review paper discusses the process of disc degeneration and the current understanding of cellular degradation in patients who present with low back pain. The role of surgical treatment for low back pain is analysed with emphasis on the proven value of spinal fusion. The interesting and novel developments of stem cell research in the treatment of low back pain are presented with special emphasis on the importance of the cartilaginous end plate and the role of IL-1 in future treatment modalities

Time-dependent Characterization of Fluid Flow into the Intervertebral Disc

The primary function of the intervertebral disc is to support large, multi-directional loads acting on the spine. The intervertebral disc has a heterogeneous structure, comprised of a gel-like nucleus pulposus (NP) and the annulus fibrosus (AF). The AF has a highly organized structure consisting of collagen fibers oriented in a criss-cross pattern in the alternating layers. Despite differences in composition and structure, water is the primary biochemical constituent of both tissues, accounting for greater than 65% of tissue’s wet weight. The water content of the intervertebral disc fluctuates throughout the day as the magnitude of compressive stress acting on the spine varies with changes in body posture, muscle activity, and external loads. The disc loses water during the day and absorbs water at night when loads are reduced.Due to the avascular nature of the intervertebral disc, cell viability and metabolism rely on the exchange of nutrients and metabolic by-products via diffusion under biochemical gradients and fluid flow modulated by diurnal loading patterns. Hence, investigating fluid flow kinematics under simulated physiological loading conditions is important for understanding healthy disc function and mechanobiology. However, there is a lack of knowledge of fluid flow behavior and recovery mechanics during low loading conditions when disc absorbs water and increases its height. Hence, this dissertation aims to fill in this gap in the literature by evaluating the time-dependent recovery mechanics and fluid flow kinematics of the healthy intervertebral disc during low loading conditions that simulate bed-rest. To achieve this, this study tested bovine bone-disc-bone motion segments under a series of creep and recovery loading conditions. Results showed that time-dependent disc recovery behavior has contributions from both inherent fluid-independent viscoelasticity and fluid-dependent poroelasticity. Intrinsic viscoelastic effects are present at short time scales, providing partial recovery of disc height within minutes of unloading before poroelastic effects come into play. Poroelastic fluid flow dominates recovery at long time scales and is largely driven by the osmotic differential between tissue and its surrounding environment. In vitro biomechanical tests on disc joints monitor changes in disc height to understand the direction, magnitude, and rate of fluid flow through the disc. However, these studies report displacements for the entire bone-disc-bone joint without the ability to identify the region-specific changes during swelling due to fluid flow. To improve our understanding of the complex fluid redistribution within the disc, the second part of this work characterized the time-dependent swelling behavior of the intervertebral disc ex situ. The first experiment monitored time-dependent changes in tissue mass to compare differences in the swelling capacities of the NP and AF explants under free swelling conditions. NP explants experienced a higher swelling rate and equilibrium swelling capacity than AF explants. Specifically, there was a 200% increase in the NP tissue mass and a 70% increase in the AF tissue mass under free swelling conditions. The second experiment used an optical, non-contact measurement method to evaluate the distribution of swelling-induced strains throughout intact discs and AF rings. Axial deformations were fixed to prevent out-of-plane motion during swelling. The first group consisted of AF rings in contact with saline at the outer periphery and the center of the annular ring. The second group included AF rings in contact with saline solution only at the outer periphery. The third group included intact discs in contact with saline at the outer periphery. Tissue swelling due to fluid flow was observed to be a slow process that strongly depends on tissue-specific biochemical properties and physical boundary constraints. For AF rings, negative circumferential strains were observed in the inner AF, while positive circumferential strains were observed in the outer AF. However, restricting fluid flow only to the outer periphery during swelling reduced the swelling capacity of the inner AF. The largest absolute radial strain was observed to be in the outer AF for Group 1 and the outer AF for Group 2. The swelling capacity of the NP was largely reduced when swelling was restricted to occur only in the radial direction or constrained by the surrounding AF. Results from intact discs showed that NP pressurization during swelling reduces peak radial strains in the AF and results in uniform strain distribution throughout the AF. Together these findings provide a better understanding of intervertebral disc mechanics and function, particularly during low loading periods when disc absorbs water and increases its volume due to swelling. In conclusion, fluid flow is a slow, time-dependent process that depends on many factors, including biochemical properties, external osmotic pressure, loading history, and boundary constraints

Nanofibrous Disc-Like Angle Ply Structure for Total Disc Replacement in a Small Animal Model

Low back pain affects 85% of the population and carries a socioeconomic price tag of $100 billion USD per year. Lumbar intervertebral disc disease is strongly implicated as a causative factor in back pain, as degeneration, which is ubiquitous in the population, leads to loss of normal spine function. For these reasons, our lab has developed disc-like angle ply structures (DAPS) for total disc replacement. These cell-seeded replacements are designed to match the natural hierarchical structure and function of the native disc and correct spinal kinematics after end-stage disc disease. In this dissertation, I describe the development of a rat caudal spine (tail) model of total disc replacement as a platform to evaluate DAPS in vivo; an external fixation system that immobilized caudal vertebrae at the site of implantation was required for DAPS retention and a radiopaque scaffold was developed to confirm intervertebral DAPS positioning. A detailed analysis of the DAPS in vitro growth trajectory was performed to select the optimum pre-culture duration before implantation. Cell-seeded DAPS were subsequently implanted in the rat tail and evaluated by histological, mechanical, and MRI analyses. DAPS successfully restored the mechanical properties of the native motion segment in compression, providing the first evidence of the efficacy of engineered disc replacements. Adaptations of the implant to the in vivo environment were identified; there was a reduction in glycosaminoglycan after implantation, structural modifications to the NP material, and no evidence of vertebral integration. In tackling the first of these issues, a pre-culture strategy that primed DAPS for the in vivo environment was developed; using a rat subcutaneous model, implant phenotype was best conserved post-implantation using a pre-culture strategy with a transient high dose of TGF-b3. Future work will address maintenance of NP structure, vertebral integration and scaling up to human sizes. In my work, the most promising finding was that DAPS replicated compressive motion segment mechanical properties after implantation supporting the idea that engineered biological disc replacement is a possibility for clinical treatment of advanced disc disease

Nerves and blood vessels in degenerated intervertebral discs are confined to physically disrupted tissue

Nerves and blood vessels are found in the peripheral annulus and endplates of healthy adult intervertebral discs. Degenerative changes can allow these vessels to grow inwards and become associated with discogenic pain, but it is not yet clear how far, and why, they grow in. Previously we have shown that physical disruption of the disc matrix, which is a defining feature of disc degeneration, creates free surfaces which lose proteoglycans and water, and so become physically and chemically conducive to cell migration. We now hypothesise that blood vessels and nerves in degenerated discs are confined to such disrupted tissue. Whole lumbar discs were obtained from 40 patients (aged 37–75 years) undergoing surgery for disc herniation, disc degeneration with spondylolisthesis or adolescent scoliosis (‘non‐degenerated’ controls). Thin (5‐μm) sections were stained with H&E and toluidine blue for semi‐quantitative assessment of blood vessels, fissures and proteoglycan loss. Ten thick (30‐μm) frozen sections from each disc were immunostained for CD31 (an endothelial cell marker), PGP 9.5 and Substance P (general and nociceptive nerve markers, respectively) and examined by confocal microscopy. Volocity image analysis software was used to calculate the cross‐sectional area of each labelled structure, and its distance from the nearest free surface (disc periphery or internal fissure). Results showed that nerves and blood vessels were confined to proteoglycan‐depleted regions of disrupted annulus. The maximum distance of any blood vessel or nerve from the nearest free surface was 888 and 247 μm, respectively. Blood vessels were greater in number, grew deeper, and occupied more area than nerves. The density of labelled blood vessels and nerves increased significantly with Pfirrmann grade of disc degeneration and with local proteoglycan loss. Analysing multiple thick sections with fluorescent markers on a confocal microscope allows reliable detection of thin filamentous structures, even within a dense matrix. We conclude that, in degenerated and herniated discs, blood vessels and nerves are confined to proteoglycan‐depleted regions of disrupted tissue, especially within annulus fissures