Changes in Intervertebral Disk Mechanical Behavior During Early Degeneration.

Intervertebral disk (IVD) degeneration is commonly described by loss of height and hydration. However, in the first stage of IVD degeneration, this loss has not yet occurred. In the current study, we use an ex vivo degeneration model to analyze the changes in IVDs mechanical behavior in the first phase of degeneration. We characterize these changes by stretched-exponential fitting, and suggest the fitted parameters as markers for early degeneration. Enzymatic degeneration of healthy lumbar caprine IVDs was induced by injecting 100 µL of Chondroïtinase ABC (Cabc) into the nucleus. A no-intervention and phosphate buffered saline (PBS) injected group were used as controls. IVDs were cultured in a bioreactor for 20 days under diurnal, simulated-physiological loading (SPL) conditions. Disk deformation was continuously monitored. Changes in disk height recovery behavior were quantified using stretched-exponential fitting. Disk height, histological sections, and water- and glycosaminoglycan (GAG)-content measurements were used as gold standards for the degenerative state. Cabc injection caused significant GAG loss from the nucleus and had detrimental effects on poro-elastic mechanical properties of the IVDs. These were progressive over time, with a propensity toward more linear recovery behavior. On histological sections, both PBS and Cabc injected IVDs showed moderate degeneration. A small GAG loss yields changes in IVD recovery behavior, which can be quantified with stretched-exponential fitting. Parameters changed significantly compared to control. Studies on disk degeneration and biomaterial engineering for degenerative disk disease (DDD) could benefit from focusing on IVD biomechanical behavior rather than height and water-content, as a marker for early disk degeneration.

Quantitative MRI in early intervertebral disc degeneration: T1rho correlates better than T2 and ADC with biomechanics, histology and matrix content.

INTRODUCTION: Low-back pain (LBP) has been correlated to the presence of intervertebral disc (IVD) degeneration on T2-weighted (T2w) MRI. It remains challenging, however, to accurately stage degenerative disc disease (DDD) based on T2w MRI and measurements of IVD height, particularly for early DDD. Several quantitative MRI techniques have been introduced to detect changes in matrix composition signifying early DDD. In this study, we correlated quantitative T2, T1rho and Apparent Diffusion Coefficient (ADC) values to disc mechanical behavior and gold standard early DDD markers in a graded degenerated lumbar IVD caprine model, to assess their potential for early DDD detection. METHODS: Lumbar caprine IVDs were injected with either 0.25 U/ml or 0.5 U/ml Chondroïtinase ABC (Cabc) to trigger early DDD-like degeneration. Injection with phosphate-buffered saline (PBS) served as control. IVDs were cultured in a bioreactor for 20 days under axial physiological loading. High-resolution 9.4 T MR images were obtained prior to intervention and after culture. Quantitative MR results were correlated to recovery behavior, histological degeneration grading, and the content of glycosaminoglycans (GAGs) and water. RESULTS: Cabc-injected IVDs showed aberrancies in biomechanics and loss of GAGs without changes in water-content. All MR sequences detected changes in matrix composition, with T1rho showing largest changes pre-to-post in the nucleus, and significantly more than T2 and ADC. Histologically, degeneration due to Cabc injection was mild. T1rho nucleus values correlated strongest with altered biomechanics, histological degeneration score, and loss of GAGs. CONCLUSIONS: T2- and T1rho quantitative MR-mapping detected early DDD changes. T1rho nucleus values correlated better than T2 and ADC with biomechanical, histological, and GAG changes. Clinical implementation of quantitative MRI, T1rho particularly, could aid in distinguishing DDD more reliably at an earlier stage in the degenerative process.

Static axial overloading primes lumbar caprine intervertebral discs for posterior herniation.

INTRODUCTION: Lumbar hernias occur mostly in the posterolateral region of IVDs and mechanical loading is an important risk factor. Studies show that dynamic and static overloading affect the nucleus and annulus of the IVD differently. We hypothesize there is also variance in the effect of overloading on the IVD's anterior, lateral and posterior annulus, which could explain the predilection of herniations in the posterolateral region. We assessed the regional mechanical and cellular responses of lumbar caprine discs to dynamic and static overloading. MATERIAL AND METHODS: IVDs (n = 125) were cultured in a bioreactor and subjected to simulated-physiological loading (SPL), high dynamic (HD), or high static (HS) overloading. The effect of loading was determined in five disc regions: nucleus, inner-annulus and anterior, lateral and posterior outer-annulus. IVD height loss and external pressure transfer during loading were measured, cell viability was mapped and quantified, and matrix integrity was assessed. RESULTS: During culture, overloaded IVDs lost a significant amount of height, yet the distribution of axial pressure remained unchanged. HD loading caused cell death and disruption of matrix in all IVD regions, whereas HS loading particularly affected cell viability and matrix integrity in the posterior region of the outer annulus. CONCLUSION: Axial overloading is detrimental to the lumbar IVD. Static overloading affects the posterior annulus more strongly, while the nucleus is relatively spared. Hence, static overloading predisposes the disc for posterior herniation. These findings could have implications for working conditions, in particular of sedentary occupations, and the design of interventions aimed at prevention and treatment of early intervertebral disc degeneration.

Bioluminescence-mediated longitudinal monitoring of adipose-derived stem cells in a large mammal ex vivo organ culture.

Recently, ex vivo three-dimensional organ culture systems have emerged to study the physiology and pathophysiology of human organs. These systems also have potential as a translational tool in tissue engineering; however, this potential is limited by our ability to longitudinally monitor the fate and action of cells used in regenerative therapies. Therefore, we investigated luciferase-mediated bioluminescence imaging (BLI) as a non-invasive technique to continuously monitor cellular behavior in ex vivo whole organ culture. Goat adipose-derived stem cells (gADSCs) were transduced with either Firefly luciferase (Fluc) or Gaussia luciferase (Gluc) reporter genes and injected in isolated goat intervertebral discs (IVD). Luciferase activity was monitored by BLI for at least seven days of culture. Additionally, possible confounders specific to avascular organ culture were investigated. Gluc imaging proved to be more suitable compared to Fluc in monitoring gADSCs in goat IVDs. We conclude that BLI is a promising tool to monitor spatial and temporal cellular behavior in ex vivo organ culture. Hence, ex vivo organ culture systems allow pre-screening and pre-validation of novel therapeutic concepts prior to in vivo large animal experimentation. Thereby, organ culture systems can reduce animal use, and improve the speed of innovation by overcoming technological, ethical and financial challenges.

Which factors prognosticate rotational instability following lumbar laminectomy?

PURPOSE: Reduced strength and stiffness of lumbar spinal motion segments following laminectomy may lead to instability. Factors that predict shear biomechanical properties of the lumbar spine were previously published. The purpose of the present study was to predict spinal torsion biomechanical properties with and without laminectomy from a total of 21 imaging parameters. METHOD: Radiographs and MRI of ten human cadaveric lumbar spines (mean age 75.5, range 59-88 years) were obtained to quantify geometry and degeneration of the motion segments. Additionally, dual X-ray absorptiometry (DXA) scans were performed to measure bone mineral content and density. Facet-sparing lumbar laminectomy was performed either on L2 or L4. Spinal motion segments were dissected (L2-L3 and L4-L5) and tested in torsion, under 1,600 N axial compression. Torsion moment to failure (TMF), early torsion stiffness (ETS, at 20-40 % TMF) and late torsion stiffness (LTS, at 60-80 % TMF) were determined and bivariate correlations with all parameters were established. For dichotomized parameters, independent-sample t tests were used. RESULTS: Univariate analyses showed that a range of geometric characteristics and disc and bone quality parameters were associated with torsion biomechanical properties of lumbar segments. Multivariate models showed that ETS, LTS and TMF could be predicted for segments without laminectomy (r (2) values 0.693, 0.610 and 0.452, respectively) and with laminectomy (r (2) values 0.952, 0.871 and 0.932, respectively), with DXA-derived measures of bone quality and quantity as the main predictors. CONCLUSIONS: Vertebral bone content and geometry, i.e. intervertebral disc width, frontal area and facet joint tropism, were found to be strong predictors of ETS, LTS and TMF following laminectomy, suggesting that these variables could predict the possible development of post-operative rotational instability following lumbar laminectomy. Proposed diagnostic parameters might aid surgical decision-making when deciding upon the use of instrumentation techniques.

Dynamic and static overloading induce early degenerative processes in caprine lumbar intervertebral discs.

Mechanical overloading of the spine is associated with low back pain and intervertebral disc (IVD) degeneration. How excessive loading elicits degenerative changes in the IVD is poorly understood. Comprehensive knowledge of the interaction between mechanical loading, cell responses and changes in the extracellular matrix of the disc is needed in order to successfully intervene in this process. The purpose of the current study was to investigate whether dynamic and static overloading affect caprine lumbar discs differently and what mechanisms lead to mechanically induced IVD degeneration. Lumbar caprine IVDs (n = 175) were cultured 7, 14 and 21 days under simulated-physiological loading (control), high dynamic or high static loading. Axial deformation and stiffness were continuously measured. Cell viability, cell density, and gene expression were assessed in the nucleus, inner- and outer annulus. The extracellular matrix (ECM) was analyzed for water, glycosaminoglycan and collagen content. IVD height loss and changes in axial deformation were gradual with dynamic and acute with static overloading. Dynamic overloading caused cell death in all IVD regions, whereas static overloading mostly affected the outer annulus. IVDs expression of catabolic and inflammation-related genes was up-regulated directly, whereas loss of water and glycosaminoglycan were significant only after 21 days. Static and dynamic overloading both induced pathological changes to caprine lumbar IVDs within 21 days. The mechanism by which they inflict biomechanical, cellular, and extracellular changes to the nucleus and annulus differed. The described cascades provide leads for the development of new pharmacological and rehabilitative therapies to halt the progression of DDD.

Torsion biomechanics of the spine following lumbar laminectomy: a human cadaver study.

PURPOSE: Lumbar laminectomy affects spinal stability in shear loading. However, the effects of laminectomy on torsion biomechanics are unknown. The purpose of this study was to investigate the effect of laminectomy on torsion stiffness and torsion strength of lumbar spinal segments following laminectomy and whether these biomechanical parameters are affected by disc degeneration and bone mineral density (BMD). METHODS: Ten human cadaveric lumbar spines were obtained (age 75.5, range 59-88). Disc degeneration (MRI) and BMD (DXA) were assessed. Disc degeneration was classified according to Pfirrmann and dichotomized in mild or severe. BMD was defined as high BMD (≥median BMD) or low BMD (

Effects of repetitive movement on range of motion and stiffness around the neutral orientation of the human lumbar spine.

In loading experiments on the lumbar spine, typically three consecutive loading cycles are applied of which the third cycle is used for analysis. The aim of this study was to investigate whether the use of ten instead of three loading cycles reduces effects of viscoelastic behavior in the assessment of range of motion (ROM) and stiffness around the neutral orientation of the human lumbar spine. To this end, twelve cadaveric human lumbar spines (L1-L5) were obtained (mean age: 76.9 years). Before testing, the spines were subjected to a compressive load of 250 N for 1 h. To each spine, ten consecutive loading cycles were applied (-4 Nm to+4 Nm) in flexion and extension (FE), lateral bending (LB) and axial rotation (AR). The ROM and stiffness within the neutral zone were calculated per motion segment (L2-L3, L3-L4 or L4-L5) from load-displacement data. It was found that the ROM increased significantly (all p<0.001) in all directions after three (FE: 0.07 degree/1.0%, LB: 0.08 degree/1.5%, and AR: 0.04 degree/1.5%) and after ten loading cycles (FE: 0.20 degree/2.9%, LB: 0.16 degree/3.3%, and AR: 0.09 degree/3.3%). Stiffness was not significantly affected, but varied considerably over cycles. Although effects were small, assessment of the tenth cycle instead of the third cycle reduces viscoelastic effects in repeated measurements of ROM, because the spine is closer to a steady state condition, while averaging over loading cycles would improve the assessment of stiffness estimates.

Simulated-physiological loading conditions preserve biological and mechanical properties of caprine lumbar intervertebral discs in ex vivo culture.

Low-back pain (LBP) is a common medical complaint and associated with high societal costs. Degeneration of the intervertebral disc (IVD) is assumed to be an important causal factor of LBP. IVDs are continuously mechanically loaded and both positive and negative effects have been attributed to different loading conditions.In order to study mechanical loading effects, degeneration-associated processes and/or potential regenerative therapies in IVDs, it is imperative to maintain the IVDs' structural integrity. While in vivo models provide comprehensive insight in IVD biology, an accompanying organ culture model can focus on a single factor, such as loading and may serve as a prescreening model to reduce life animal testing. In the current study we examined the feasibility of organ culture of caprine lumbar discs, with the hypothesis that a simulated-physiological load will optimally preserve IVD properties.Lumbar caprine IVDs (n = 175) were cultured in a bioreactor up to 21 days either without load, low dynamic load (LDL), or with simulated-physiological load (SPL). IVD stiffness was calculated from measurements of IVD loading and displacement. IVD nucleus, inner- and outer annulus were assessed for cell viability, cell density and gene expression. The extracellular matrix (ECM) was analyzed for water, glycosaminoglycan and total collagen content.IVD biomechanical properties did not change significantly with loading conditions. With SPL, cell viability, cell density and gene expression were preserved up to 21 days. Both unloaded and LDL resulted in decreased cell viability, cell density and significant changes in gene expression, yet no differences in ECM content were observed in any group.In conclusion, simulated-physiological loading preserved the native properties of caprine IVDs during a 21-day culture period. The characterization of caprine IVD response to culture in the LDCS under SPL conditions paves the way for controlled analysis of degeneration- and regeneration-associated processes in the future.

Which factors prognosticate spinal instability following lumbar laminectomy?

PURPOSE: Reduced strength and shear stiffness (SS) of lumbar motion segments following laminectomy may lead to instability. The purpose of the present study was to assess a broad range of parameters as potential predictors of shear biomechanical properties of the lumbar spine. METHODS: Radiographs and MRI of all lumbar spines were obtained to classify geometry and degeneration of the motion segments. Additionally, dual X-ray absorptiometry (DXA) scans were performed to measure bone mineral content and density (BMC and BMD). Facet sparing lumbar laminectomy was performed either on L2 or L4, in 10 human cadaveric lumbar spines (mean age 72.1 years, range 53-89 years). Spinal motion segments were dissected (L2-L3 and L4-L5) and tested in shear, under simultaneously loading with 1600 N axial compression. Shear stiffness, shear yield force (SYF) and shear force to failure (SFF) were determined and statistical correlations with all parameters were established. RESULTS: Following laminectomy, SS, SYF, and SFF declined (by respectively 24, 41, and 44%). For segments with laminectomy, SS was significantly correlated with intervertebral disc degeneration and facet joint degeneration (Pfirrmann: r = 0.64; Griffith: r = 0.70; Lane: r = 0.73 and Pathria: r = 0.64), SYF was correlated with intervertebral disc geometry (r = 0.66 for length; r = 0.66 for surface and r = 0.68 for volume), BMC (r = 0.65) and frontal area (r = 0.75), and SFF was correlated with disc length (r = 0.73) and BMC (r = 0.81). For untreated segments, SS was significantly correlated with facet joint tropism (r = 0.71), SYF was correlated with pedicle geometry (r = 0.83), and SFF was correlated with BMC (r = 0.85), BMD (r = 0.75) and frontal area (r = 0.75). SS, SYF and SFF could be predicted for segments with laminectomy (r (2) values respectively: 0.53, 0.81 and 0.77) and without laminectomy (r (2) value respectively: 0.50, 0.83 and 0.83). CONCLUSIONS: Significant loss of strength and SS are predicted by BMC, BMD, intervertebral disc geometry and degenerative parameters, suggesting that low BMC or BMD, small intervertebral discs and absence of osteophytes could predict the possible development of post-operative instability following lumbar laminectomy.