A novel in-vitro model of intervertebral disc degeneration using hyperphysiological loading.

Intervertebral disc (IVD) degeneration includes changes in tissue biomechanics, physical attributes, biochemical composition, disc microstructure, and cellularity, which can all affect the normal function of the IVD, and ultimately may lead to pain. The purpose of this research was to develop an in-vitro model of degeneration that includes the evaluation of physical, biomechanical, and structural parameters, and that does so over several load/recovery periods. Hyperphysiological loading was used as the degenerative initiator with three experimental groups employed using bovine coccygeal IVD specimens: Control; Single-Overload; and Double-Overload. An equilibrium stage comprising a static load followed by two load/recovery periods was followed by six further load/recovery periods. In the Control group all load/recovery periods were the same, comprising physiological cyclic loading. The overload groups differed in that hyperphysiological loading was applied during the 4th loading period (Single-Overload), or the 4th and 5th loading period (Double-Overload). Overloading led to a significant reduction in disc height compared to the Control group, which was not recovered in subsequent physiological load/recovery periods. However, there were no significant changes in stiffness. Overloading also led to significantly more microstructural damage compared to the Control group. Taking all outcome measures into account, the overload groups were evaluated as replicating clinically relevant aspects of moderate IVD degeneration. Further research into a potential dose-effect, and how more severe degeneration can be replicated would provide a model with the potential to evaluate new treatments and interventions for different stages of IVD degeneration.

In-vitro models of disc degeneration - A review of methods and clinical relevance.

The intervertebral disc (IVD) provides flexibility, acts as a shock absorber, and transmits load. Degeneration of the IVD includes alterations in the biomechanics, extracellular matrix (ECM), and cellular activity. These changes are not always perceived, however, IVD degeneration can lead to severe health problems including long-term disability. To understand the pathogenesis of IVD degeneration and suitable testing methods for emerging treatments and therapies, this review documents in-vitro models of IVD degeneration including physical disruption, hyperphysiological loading, ECM degradation by enzyme digestion, or a combination of these methods. This paper reviews and critically analyses the models of degeneration published since the year 2000 in either in human or animal specimens. The results are categorised in terms of the IVD biomechanics, physical attributes, ECM composition, tissue damage and cellularity to evaluate the models with respect to natural human degeneration, and to provide recommendations for clinically relevant models for the various stages of degeneration. There is no one model that replicates the wide range of degenerative changes that occur as part of normal degeneration. However, cyclic overloading replicates many aspects of degeneration, with the advantage of a dose-response allowing the tuning of damage initiated. Models of severe degeneration are currently lacking, but there is potential that combining cyclic overloading and enzymatic digestion will provide model that closely resembles human IVD degeneration. This will provide an effective way to investigate the effects of severe degeneration, and the evaluation of treatments for the IVD, which would generally be indicated at this advanced stage of degeneration.

Micro-mechanical damage of needle puncture on bovine annulus fibrosus fibrils studied using polarization-resolved Second Harmonic Generation(P-SHG) microscopy.

Needle injection has been widely used in spinal therapeutic or diagnostic processes, such as discography. The use of needles has been suspected in causing mild disc degeneration which can lead to long-term back pain. However, the localised microscopic damage caused by needles has not been well studied. The local progressive damage on a microscopic level caused by needle punctures on the surface of bovine annulus fibrosus was investigated. Four different sizes of needle were used for the puncture and twenty-nine bovine intervertebral discs were studied. Polarization-resolved second harmonic generation and fluorescent microscopy were used to study the local microscopic structural changes in collagen and cell nuclei due to needle damage. Repeated 70 cyclic loadings at ±5% of axial strain were applied after the needle puncture in order to assess progressive damage caused by the needle. Puncture damage on annulus fibrosus were observed either collagen fibre bundles being pushed aside, being cut through or combination of both with part being lift or pushed in. The progressive damage was found less relevant to the needle size and more progressive damage was only observed using the larger needle. Two distinct populations of collagen, in which one was relatively more organised than the other population, were observed especially after the puncture from skewed distribution of polarization-SHG analysis. Cell shape was found rounder near the puncture site where collagen fibres were damaged.

Computer-aided detection in musculoskeletal projection radiography: A systematic review.

OBJECTIVES: To investigated the accuracy of computer-aided detection (CAD) software in musculoskeletal projection radiography via a systematic review. KEY FINDINGS: Following selection screening, eligible studies were assessed for bias, and had their study characteristics extracted resulting in 22 studies being included. Of these 22 three studies had tested their CAD software in a clinical setting; the first study investigated vertebral fractures, reporting a sensitivity score of 69.3% with CAD, compared to 59.8% sensitivity without CAD. The second study tested dental caries diagnosis producing a sensitivity score of 68.8% and specificity of 94.1% with CAD, compared to sensitivity of 39.3% and specificity of 96.7% without CAD. The third indicated osteoporotic cases based on CAD, resulting in 100% sensitivity and 81.3% specificity. CONCLUSION: The current evidence reported shows a lack of development into the clinical testing phase; however the research does show future promise in the variation of different CAD systems.

Sex Differences in the Associations between L-Arginine Pathway Metabolites, Skeletal Muscle Mass and Function, and their Responses to Resistance Exercise, in Old Age.

OBJECTIVES: The current study was designed to explore the associations between L-arginine metabolites and muscle mass and function in old age, which are largely unknown. DESIGN: The study used a randomised, double-blind, placebo-controlled design. SETTING: The study was carried out in a laboratory setting. PARTICIPANTS: 50 healthy older adults [median age 70 years (IQR 67-73); 27 males]. INTERVENTION: Participants undertook an 18-week resistance exercise program, and a nutritional intervention (fish oil vs. placebo). MEASUREMENTS: Serum homoarginine, ornithine, citrulline, asymmetric dimethylarginine (ADMA), NG-monomethyl-L-arginine (L-NMMA), and symmetric dimethylarginine (SDMA), maximal voluntary contraction (MVC) and isokinetic torque of the knee extensors at 30° s-1 (MIT), muscle cross sectional area (MCSA) and quality (MQ) were measured at baseline and after the intervention. RESULTS: No significant exercise-induced changes were observed in metabolite concentrations. There were significant sex differences in the associations between metabolites and muscle parameters. After adjusting for age, glomerular filtration rate and fish oil intervention, citrulline (P=0.002) and ornithine (P=0.022) were negatively associated with MCSA at baseline in males but not females. However, baseline citrulline was negatively correlated with exercise-induced changes in MVC (P=0.043) and MQ (P=0.026) amongst females. Furthermore, amongst males, baseline homoarginine was positively associated with exercise-induced changes in MVC (P=0.026), ADMA was negatively associated with changes in MIT (P=0.026), L-NMMA (p=0.048) and ornithine (P<0.001) were both positively associated with changes in MCSA, and ornithine was negatively associated with changes in MQ (P=0.039). CONCLUSION: Therefore, barring citrulline, there are significant sex differences in the associations between L-arginine metabolites and muscle mass and function in healthy older adults. These metabolites might enhance sarcopenia risk stratification, and the success of exercise programs, in old age.

Effects of sex, age, body height and body weight on spinal loads: Sensitivity analyses in a subject-specific trunk musculoskeletal model.

Subject-specific parameters influence spinal loads and the risk of back disorders but their relative effects are not well understood. The objective of this study is to investigate the effects of changes in age (35-60 years), sex (male, female), body height (BH: 150-190cm) and body weight (BW: 50-120kg) on spinal loads in a full-factorial simulation using a personalized (spine kinematics, geometry, musculature and passive properties) kinematics driven musculoskeletal trunk finite element model. Segmental weight distribution (magnitude and location along the trunk) was estimated by a novel technique to accurately represent obesity. Five symmetric sagittal loading conditions were considered, and main effect plots and analyses of variance were employed to identify influential parameters. In all 5 tasks simulated, BW (98.9% in compression and 96.1% in shear) had the greatest effect on spinal loads at the L4-L5 and L5-S1 levels followed by sex (0.7% in compression and 2.1% in shear), BH (0.4% in compression and 1.5% in shear) and finally age (<5.4%). At identical BH and BW, spinal loads in females were slightly greater than those in males by ~4.7% in compression and ~8.7% in shear. In tasks with no loads in hands, BW-normalized spinal loads further increased with BW highlighting the exponential increase in spinal loads with BW that indicates the greater risk of back disorders especially in obese individuals. Uneven distribution of weight in obese subjects, with more BW placed at the lower trunk, further (though slightly <7.5%) increased spinal loads.

Phase II study of single-agent panitumumab in patients with incurable cutaneous squamous cell carcinoma.

BACKGROUND: Although advanced cutaneous squamous cell carcinoma (CSCC) is quite common, there are few prospective trials regarding its optimal management. This study evaluated the efficacy and safety of single-agent panitumumab in the treatment of patients with CSCC not suitable for local therapy. PATIENTS AND METHODS: Sixteen patients received single-agent panitumumab at a dose of 6 mg/kg repeated every 2 weeks for a minimum of three cycles and continued until progression, a maximum of nine cycles or dose-limiting toxicity. The primary end point was the best overall response rate (ORR) as assessed by Response Evaluation Criteria in Solid Tumours (RECIST version 1.1) criteria. Secondary end points included evaluation of safety, toxicity and progression-free survival (PFS). RESULTS: Between May 2010 and May 2012, 16 patients were recruited. Fourteen patients were male and the median age was 68 years. Fifteen patients had locoregionally advanced or recurrent disease with 14 patients receiving previous radiotherapy and 7 receiving previous cytotoxic chemotherapy. The best ORR [partial (PR) or complete response (CR)] was 31% (3/16 PR, 2/16 CR) with a further 6 of 16 patients achieving SD. The median PFS and overall survival were 8 and 11 months respectively. Grade 3 or 4 events were observed in five patients (four being skin toxicity) with one patient ceasing due to skin toxicity. With a median follow-up of 24 months, 10 patients died due to progressive disease, 6 are alive, one patient with no evidence of disease at the time of analysis. CONCLUSIONS: Single-agent panitumumab is safe and effective in the management of patients with advanced CSCC even in a previously extensively pre-treated cohort.

The reliability of (31)P-MRS and NIRS measurements of spinal muscle function.

While phosphorous magnetic resonance spectroscopy ((31)P-MRS) and near-infrared spectroscopy (NIRS) provide methods for measuring spinal muscle function non-invasively, their reliability is not established. The aim of this study was assess the reliability (ICC) and error magnitude (CV%) of measurements of muscle phosphocreatine (PCr), tissue oxygenation index (TOI) and muscle deoxyhaemoglobin (HHb) acquired during fatigue and in recovery after 24 s of exercise in the lumbar muscles. 10 healthy participants (19-25 years, 5 male, 5 female) performed exercise that involved holding the upper body unsupported in slight extension until fatigue and then, after 30 min of rest, for repeated bursts of 24 s. ICCs indicated good to excellent reliability of baseline measures (TOI: 0.75) and of amplitude changes during fatigue (PCr: 0.73, TOI: 0.69, HHb: 0.80) and recovery (HHb: 0.96), and poor to fair reliability for time constants describing rates of change during fatigue (PCr: 0.11) and recovery (PCr: 0.31, HHb: 0.47). CV% indicated varying relative measurement error across baseline measures (TOI: 5%), amplitude changes during fatigue (PCr: 7%, TOI: 38%, HHb: 31%) and recovery (HHb: 31%), and in time constants for fatigue (PCr: 39%) and recovery (PCr: 20%, HHb: 37%). The results suggested that reliability would be sufficient for future studies on spinal muscle function, but that measurement error may be too large to evaluate individuals.

Compression or tension? The stress distribution in the proximal femur.

BACKGROUND: Questions regarding the distribution of stress in the proximal human femur have never been adequately resolved. Traditionally, by considering the femur in isolation, it has been believed that the effect of body weight on the projecting neck and head places the superior aspect of the neck in tension. A minority view has proposed that this region is in compression because of muscular forces pulling the femur into the pelvis. Little has been done to study stress distributions in the proximal femur. We hypothesise that under physiological loading the majority of the proximal femur is in compression and that the internal trabecular structure functions as an arch, transferring compressive stresses to the femoral shaft. METHODS: To demonstrate the principle, we have developed a 2D finite element model of the femur in which body weight, a representation of the pelvis, and ligamentous forces were included. The regions of higher trabecular bone density in the proximal femur (the principal trabecular systems) were assigned a higher modulus than the surrounding trabecular bone. Two-legged and one-legged stances, the latter including an abductor force, were investigated. RESULTS: The inclusion of ligamentous forces in two-legged stance generated compressive stresses in the proximal femur. The increased modulus in areas of greater structural density focuses the stresses through the arch-like internal structure. Including an abductor muscle force in simulated one-legged stance also produced compression, but with a different distribution. CONCLUSION: This 2D model shows, in principle, that including ligamentous and muscular forces has the effect of generating compressive stresses across most of the proximal femur. The arch-like trabecular structure transmits the compressive loads to the shaft. The greater strength of bone in compression than in tension is then used to advantage. These results support the hypothesis presented. If correct, a better understanding of the stress distribution in the proximal femur may lead to improvements in prosthetic devices and an appreciation of the effects of various surgical procedures affecting load transmission across the hip.

A mechanism for balancing the human body on the hips.

A mechanism for the postural balance of the human torso over the hip joints is reported that does not appear to have been fully recognised. The centre of gravity of the torso is usually considered poised above the hips. This is in a state of unstable equilibrium. Here, we propose that body weight acting through the sacrum is counter-balanced by tension in the ilio-femoral ligament; the hips acting as the fulcrum. This places the torso in stable equilibrium and means that balance may be maintained with minimal muscular forces. The joint reaction force due to this mechanism in a two-legged stance is then of the order of the body-weight on each hip. An implication is that capsular ligaments are important and consideration should be given to retaining or restoring them as much as possible in hip arthroplasty.

Influence of fibril taper on the function of collagen to reinforce extracellular matrix.

Collagen fibrils provide tensile reinforcement for extracellular matrix. In at least some tissues, the fibrils have a paraboloidal taper at their ends. The purpose of this paper is to determine the implications of this taper for the function of collagen fibrils. When a tissue is subjected to low mechanical forces, stress will be transferred to the fibrils elastically. This process was modelled using finite element analysis because there is no analytical theory for elastic stress transfer to a non-cylindrical fibril. When the tissue is subjected to higher mechanical forces, stress will be transferred plastically. This process was modelled analytically. For both elastic and plastic stress transfer, a paraboloidal taper leads to a more uniform distribution of axial tensile stress along the fibril than would be generated if it were cylindrical. The tapered fibril requires half the volume of collagen than a cylindrical fibril of the same length and the stress is shared more evenly along its length. It is also less likely to fracture than a cylindrical fibril of the same length in a tissue subjected to the same mechanical force.

Short communication: fused deposition models from CT scans.

Fused deposition modelling (FDM) is a new method for rapid prototyping, a technique that produces models of objects from computer files. The most commonly used rapid prototyping technique for medical applications is stereolithography, but FDM has several potential advantages. This paper is concerned with the accuracy of an FDM model of a sheep lumbar vertebra using data from a CT scan. The model and the original vertebra were compared by making measurements with vernier callipers and by laser scanning. Visually, the model reproduced the features of the original object; this conclusion was supported by a comparison of the laser scans. Discrepancies in measurements were comparable with those of models produced using other rapid prototyping techniques, demonstrating that FDM is a viable method for making models for clinical use.

Rheological properties of poly(2-hydroxyethyl methacrylate) (pHEMA) as a function of water content and deformation frequency.

Poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels have been used, or suggested for use, in a wide range of biomedical applications. In many of these applications, the mechanical properties of the gel are important for its proper functioning. These properties are influenced by a number of factors, including water content. In this study the storage and loss shear moduli were measured as a function of frequency for gels with water contents ranging from 22% to 48% at a temperature of 37 degrees C. At low frequencies and high water contents, deformation frequency had little effect. However, at higher frequencies and lower water contents, both moduli increased markedly with increasing frequency. This can be explained by the gels approaching a glass transition. The curves describing the behavior of each gel were combined to form a master curve, using a method analogous to the time-temperature superposition principle. This master curve can be used to predict the shear moduli for gels with a wide range of water contents and loading frequencies. For example, for a gel with a water content of 47.8% (as a percentage of the mass of gel), the curve provides shear moduli values over a frequency range of 10(-2)-10(4) Hz.

Thermal analysis of poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels.

The influence of water on the physical properties of a hydrogel is important for understanding natural tissues and in designing synthetic materials to replace them. In this study, poly (2-hydroxyethyl methacrylate) (pHEMA) was used as a model system to understand how water interacts with the polymer of a hydrogel. Thermal analysis methods (thermogravimetric analysis coupled to mass spectrometry and differential scanning calorimetry) were used to determine: (i) the total water content of pHEMA gels; (ii) how this water was lost during heating; (iii) the relationship between water content of the gel and its glass transition temperature; and (iv) the behavior of the water in the gel on cooling. Previous researchers have invoked various models to describe the organization of water in a hydrogel. In this study, the simplest model which could explain all of the results from the different thermal analysis techniques was one which consisted of three classes of water: (i) hydration water in close proximity to the polymer; (ii) interstitial water in regions or cavities surrounded by polymer chains; and (iii) bulk water.

Sheep lumbar intervertebral discs as models for human discs.

OBJECTIVE: To determine the water content, collagen content and collagen orientation angle in different regions of sheep lumbar discs. DESIGN: A laboratory study of sheep discs obtained from an abattoir. METHODS: A total of 21 sheep lumbar discs were obtained from three lumbar spines. Water content was determined by oven drying (60 degrees C) to constant mass. Collagen content was determined by hydroxyproline analysis. Fibre orientation angles were determined by X-ray diffraction. RESULTS: Water content increased from 74% of total tissue mass in the outer annulus, to 82% in the inner annulus, to 86% in the nucleus. Collagen content decreased from 30% of total tissue mass in the outer region to 20% in the inner region of the anterior and lateral annulus; it was 16% in the posterior annulus. The orientation angle of the collagen fibres decreased from 59 degrees in the outer region to 56 degrees in the inner region of the anterior and lateral annulus; it was 51 degrees in the posterior annulus. CONCLUSIONS: Sheep lumbar intervertebral discs provide a reasonable model for human lumbar intervertebral discs. RELEVANCE: Sheep lumbar discs have been used to investigate the effects of removing and replacing the nucleus. These studies indicate that removal of nucleus may lead to further disc degeneration and indicate the material properties required for an implant material. The relevance of these previous studies is increased if human and sheep lumbar discs have a similar composition and structure.

Replacing the nucleus pulposus of the intervertebral disc.

OBJECTIVE: To determine whether replacement of the nucleus with a synthetic material would prevent the effects of nucleus removal. DESIGN: Laboratory experiments on excised tissues and a finite element model. BACKGROUND: Removal of the nucleus from the intervertebral disc causes the inner margins of the annulus to bulge inwards, instead of outwards, during compression. This may cause the annulus to degenerate further. METHODS: Video recordings of sheep discs, sectioned in the sagittal plane, were obtained during compression in a materials testing machine; the cut face of the disc was sealed with a Perspex window. Experiments were repeated with the nucleus removed and then replaced by a synthetic implant. A finite element model of an intact disc was also used to investigate the effect of nucleus replacement. RESULTS: When the nucleus of sectioned discs was replaced with the polymer materials, the inward bulging of the annulus was not observed. The predictions from the finite element model of the intact disc were consistent with this result. CONCLUSIONS: Replacement of the nucleus with a synthetic material can prevent the changes in annulus behaviour that result from removal of the nucleus. RELEVANCE: A suitable implant to replace the nucleus after surgical removal may help prevent inward bulging of the inner layers of the annulus.

The effect of partial removal of the nucleus pulposus from the intervertebral disc on the response of the human annulus fibrosus to compression.

OBJECTIVE: To determine how partial removal of the nucleus changes the response of the annulus to compression. DESIGN: The deformation of the annulus in the mid-sagittal plane, during compression, was determined from digital video images. BACKGROUND: Several studies have shown that removal of the nucleus changes the external behaviour of the intervertebral disc, but few studies have investigated changes to internal behaviour. METHODS: Six frozen human lumbar discs were bisected in the sagittal plane to produce 12 specimens. The cut surfaces were marked with seven dots of Alcian blue stain. The specimens were sealed, enabling their internal structure to be viewed directly by a digital video recording system, and thawed. The video system recorded the response of each specimen as it was compressed by up to 1.8 mm at a rate of 0.2 mm s(-1). The displacements of the Alcian blue marks were measured using an image analysis program. Magnetic resonance imaging was used to investigate the validity of this technique. RESULTS: Partial removal of the nucleus changed the way that the disc deformed under compression. A highly significant change in direction of movement was seen in the inner posterior region of the annulus. CONCLUSIONS: Partial removal of the nucleus changes the response of the annulus to compression. RELEVANCE: Partial denucleation of the human intervertebral disc is shown to change the direction of bulging of the inner annulus when the disc is compressed. Increases in shear stress, arising from these changes, may lead to further disc degeneration in the form of circumferential tears.

Replacing the nucleus pulposus of the intervertebral disk: prediction of suitable properties of a replacement material using finite element analysis.

An axisymmetric finite element model of a human lumbar disk was developed to investigate the properties required of an implant to replace the nucleus pulposus. In the intact disk, the nucleus was modeled as a fluid, and the annulus as an elastic solid. The Young's modulus of the annulus was determined empirically by matching model predictions to experimental results. The model was checked for sensitivity to the input parameter values and found to give reasonable behavior. The model predicted that removal of the nucleus would change the response of the annulus to compression. This prediction was consistent with experimental results, thus validating the model. Implants to fill the cavity produced by nucleus removal were modeled as elastic solids. The Poisson's ratio was fixed at 0.49, and the Young's modulus was varied from 0.5 to 100 MPa. Two sizes of implant were considered: full size (filling the cavity) and small size (smaller than the cavity). The model predicted that a full size implant would reverse the changes to annulus behavior, but a smaller implant would not. By comparing the stress distribution in the annulus, the ideal Young's modulus was predicted to be approximately 3 MPa. These predictions have implications for current nucleus implant designs.