Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.     

Infrared attenuated total reflection spectroscopic surface analysis of bovine‐tail intervertebral discs after UV‐light‐activated riboflavin‐induced collagen cross‐linking

The tensile strength of the intervertebral disc (IVD) is mainly maintained by collagen cross‐links. Loss of collagen cross‐linking combined with other age‐related degenerative processes contributes to tissue weakening, biomechanical failure, disc herniation and pain. Exogenous collagen cross‐linking has been identified as an effective therapeutic approach for restoring IVD tensile strength. The current state‐of‐the‐art method to assess the extent of collagen cross‐linking in tissues requires destructive procedures and high‐performance liquid chromatography. In this study, we investigated the utility of infrared attenuated total reflection (IR‐ATR) spectroscopy as a nondestructive analytical strategy to rapidly evaluate the extent of UV‐light‐activated riboflavin (B2)‐induced collagen cross‐linking in bovine IVD samples. Thirty‐five fresh bovine‐tail IVD samples were equally divided into five treatment groups: (a) untreated, (b) cell culture medium Dulbecco's Modified Eagle's Medium only, (c) B2 only, (d) UV‐light only and (e) UV‐light‐B2. A total of 674 measurements have been acquired, and were analyzed via partial least squares discriminant analysis. This classification scheme unambiguously identified individual classes with a sensitivity >91% and specificity >92%. The obtained results demonstrate that IR‐ATR spectroscopy reliably differentiates between different treatment categories, and promises an excellent tool for potential in vivo, nondestructive and real‐time assessment of exogenous IVD cross‐linking.     

Does the correlation between schmorl's nodes and vertebral morphology extend into the lumbar spine?

Schmorl's nodes are depressions on vertebrae due to herniation of the nucleus pulposus of the intervertebral disc into the vertebral body. This study provides an extension of our previous study which analyzed the shape of the lower thoracic spine and found that vertebral morphology was associated with the presence of Schmorl's nodes. Ninety adult individuals from the late Medieval site of Fishergate House, York, and the Post‐Medieval site of Coach Lane, North Shields, Tyne and Wear, England, were analysed using 2D geometric morphometrics to identify possible relationships between vertebral morphology and Schmorl's nodes at the thoraco‐lumbar junction and in the lumbar spine. A significant correlation was found between vertebral shape and the presence of Schmorl's nodes in the twelfth thoracic vertebrae and the first to third lumbar vertebrae. The findings corroborate previous studies and suggest that vertebral shape may be an important factor in spinal health. It is hypothesized that the pedicle shape of affected vertebrae may not provide adequate structural support for the vertebral bodies, resulting in vertical disc herniation. Am J Phys Anthropol 157:526–534, 2015. © 2015 Wiley Periodicals, Inc.     

Nucleus implant parameters significantly change the compressive stiffness of the human lumbar intervertebral disc

Nucleus replacement by a synthetic material is a recent trend for treatment of lower back pain. Hydrogel nucleus implants were prepared with variations in implant modulus, height, and diameter Human lumbar intervertebral discs (IVDs) were tested in compression for intact, denucleated, and implanted condition. Implantation of nucleus implants with different material and geometric parameters into a denucleated IVD significantly altered the IVD compressive stiffness. Variations in the nucleus implant parameters significantly change the compressive stiffness of the human lumbar IVD. Implant geometrical variations were more effective than those of implant modulus variations in the range examined.     

Personalised mechanical properties of scoliotic vertebrae determined in vivo using tomodensitometry

This in vivo study investigated the mechanical properties of apical scoliotic vertebrae using computed tomography (CT) and finite element (FE) meshing. CT examination was performed on seven scoliotic girls. FE meshing of each vertebral body allowed automatic mapping of the CT scan and the visualisation of the bone density distribution. Centroids and mass centres were compared to analyse the mechanical properties distribution. Compared to the centroid, the mass centre migrated into the concavity of the curvature. The three vertebrae of a same patient had the same body migration behaviour because they were located at the curvature apex. This observation was verified in the coronal plane, but not in the sagittal plane. These results represent new data over few geometrical analyses of scoliotic vertebrae. Same in vivo personalisation of mechanical properties should be performed on intervertebral discs or ligaments to personalise stiffness properties of the spine for the biomechanical modelling of human torso. Moreover, do this mechanical deformation of scoliotic vertebrae, that appears before the vertebral wedging, could be a predictive tool in scoliosis treatment?     

Caudal vertebral body articular surface morphology correlates with functional tail use in anthropoid primates

Prehensile tails, capable of suspending the entire body weight of an animal, have evolved in parallel in New World monkeys (Platyrrhini): once in the Atelinae (Alouatta, Ateles, Brachyteles, Lagothrix), and once in the Cebinae (Cebus, Sapajus). Structurally, the prehensile tails of atelines and cebines share morphological features that distinguish them from nonprehensile tails, including longer proximal tail regions, well‐developed hemal processes, robust caudal vertebrae resistant to higher torsional and bending stresses, and caudal musculature capable of producing higher contractile forces. The functional significance of shape variation in the articular surfaces of caudal vertebral bodies, however, is relatively less well understood. Given that tail use differs considerably among prehensile and nonprehensile anthropoids, it is reasonable to predict that caudal vertebral body articular surface area and shape will respond to use‐specific patterns of mechanical loading. We examine the potential for intervertebral articular surface contour curvature and relative surface area to discriminate between prehensile‐tailed and nonprehensile‐tailed platyrrhines and cercopithecoids. The proximal and distal intervertebral articular surfaces of the first (Ca1), transitional and longest caudal vertebrae were examined for individuals representing 10 anthropoid taxa with differential patterns of tail‐use. Study results reveal significant morphological differences consistent with the functional demands of unique patterns of tail use for all vertebral elements sampled. Prehensile‐tailed platyrrhines that more frequently use their tails in suspension (atelines) had significantly larger and more convex intervertebral articular surfaces than all nonprehensile‐tailed anthropoids examined here, although the intervertebral articular surface contour curvatures of large, terrestrial cercopithecoids (i.e., Papio sp.) converge on the ateline condition. Prehensile‐tailed platyrrhines that more often use their tails in tripodal bracing postures (cebines) are morphologically intermediate between atelines and nonprehensile tailed anthropoids. J. Morphol. 275:1300–1311, 2014. © 2014 Wiley Periodicals, Inc.     

Personalised Mechanical Properties of Scoliotic Vertebrae Determined In Vivo Using Tomodensitometry

This in vivo study investigated the mechanical properties of apical scoliotic vertebrae using computed tomography (CT) and finite element (FE) meshing. CT examination was performed on seven scoliotic girls. FE meshing of each vertebral body allowed automatic mapping of the CT scan and the visualisation of the bone density distribution. Centroids and mass centres were compared to analyse the mechanical properties distribution. Compared to the centroid, the mass centre migrated into the concavity of the curvature. The three vertebrae of a same patient had the same body migration behaviour because they were located at the curvature apex. This observation was verified in the coronal plane, but not in the sagittal plane. These results represent new data over few geometrical analyses of scoliotic vertebrae. Same in vivo personalisation of mechanical properties should be performed on intervertebral discs or ligaments to personalise stiffness properties of the spine for the biomechanical modelling of human torso. Moreover, do this mechanical deformation of scoliotic vertebrae, that appears before the vertebral wedging, could be a predictive tool in scoliosis treatment?     

Loss and re-adaptation of lumbar intervertebral disc water signal intensity after prolonged bedrest

The adaptation and re-adaptation process of the intervertebral disc (IVD) to prolonged bedrest is important for understanding IVD physiology and IVD herniations in astronauts. Little information is available on changes in IVD composition. In this study, 24 male subjects underwent 60-day bedrest and In/Out Phase magnetic resonance imaging sequences were performed to evaluate IVD shape and water signal intensity. Scanning was performed before bedrest (baseline), twice during bedrest, and three, six and twenty-four months after bedrest. Area, signal intensity, average height, and anteroposterior diameter of the lumbar L3/4 and L4/5 IVDs were measured. At the end of bedrest, disc height and area were significantly increased with no change in water signal intensity. After bedrest, we observed reduced IVD signal intensity three months (p=0.004 versus baseline), six months (p=0.003 versus baseline), but not twenty-four months (p=0.25 versus baseline) post-bedrest. At these same time points post-bedrest, IVD height and area remained increased. The reduced lumbar IVD water signal intensity in the first months after bedrest implies a reduction of glycosaminoglycans and/or free water in the IVD. Subsequently, at two years after bedrest, IVD hydration status returned towards pre-bedrest levels, suggesting a gradual, but slow, re-adaptation process of the IVD after prolonged bedrest.     

Finite element simulation of an artificial intervertebral disk using fiber reinforced laminated composite model

Degeneration of intervertebral disk (IVD) has been increased in recent years. The lumbar herniation can be cured using conservative and surgical procedures. Surgery is considered after failure of conservative treatment. Partial discectomy, fusion, and total disk replacement (TDR) are also common surgical treatments for degenerative disk disease. However, due to limitations and disadvantages of the current treatments, many studies have been carried out to approach the best design of mimicking natural disk. Recently, a new method of TDRs has been introduced using nature deformation of IVD by reinforced fibers of annulus fibrosis. Nonetheless, owing to limitations of experimental works on the human body, numerical studies of IVD may help to understand load transfer and biomechanical properties within the disks with reinforced fibers. In this study, a three-dimensional (3D) finite element model of the L2-L3 disk vertebrae unit with 12 vertical fibers embedded into annulus fibrosis was constructed. The IVD was subjected to compressive force, bending moment, and axial torsion. The most important parameters of disk failures were compared to that of experimental data. The results showed that the addition of reinforced fibers into the disk invokes a significant decrease of stress in the nucleus and annulus. The findings of this study may have implications not only for developing IVDs with reinforced fibers but also for the application of fiber reinforced IVD in orthopedics surgeries as a suitable implant.     

Regional variation in morphology of vertebral centra and intervertebral joints in striped bass,Morone saxatilis

The vertebral column of fishes has traditionally been divided into just two distinct regions, abdominal and caudal. Recently, however, developmental, morphological, and mechanical investigations have brought this traditional regionalization scheme into question. Alternative regionalization schema advocate the division of the abdominal vertebrae into cervical, abdominal, and in some cases, transitional regions. Here, we investigate regional variation at the level of the vertebrae and intervertebral joint (IVJ) tissues in the striped bass, Morone saxatilis. We use gross dissection, histology, and polarized light imaging to quantify vertebral height, width, length, IVJ length, IVJ tissue volume and cross‐sectional area, and vertical septum fiber populations, and angles of insertion. Our results reveal regional differences between the first four (most rostral) abdominal vertebrae and IVJs and the next six abdominal vertebrae and IVJs, supporting the recognition of a distinct cervical region. We found significant variation in vertebral length, width, and height from cranial to caudal. In addition, we see a significant decline in the volume of notochordal cells and the cross‐sectional area of the fibrous sheath from cranial to caudal. Further, polarized light imaging revealed four distinct fiber populations within the vertical septum in the cervical and abdominal regions in contrast with just one fiber population found in the caudal region. Measurement of the insertion angles of these fiber populations revealed significant differences between the cervical and abdominal regions. Differences in vertebral, IVJ, and vertical septum morphology all predict greater range of motion and decreased stiffness in the caudal region of the fish compared with the cervical and abdominal regions. J. Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

Influences of Endplate Removal and Bone Mineral Density on the Biomechanical Properties of Lumbar Spine

Purpose

To investigate (1) effects of endplate removal and bone mineral density (BMD) on biomechanical properties of lumbar vertebrae (2) whether the distributions of mechanical strength and stiffness of endplate are affected by BMD.

Methods

A total of thirty-one lumbar spines (L1-L5) collected from fresh cadavers were used in this study. Bone density was measured using lateral DEXA scans and parts of samples were performed with partial or entire endplate removal. All the specimens were divided into three BMD groups. According to endplate integrity of the lumbar vertebrae, each BMD group was then divided into three subgroups: subgroup A: intact endplate; subgroup B: central region of endplate removal; subgroup C: entire endplate removal. The axial compression test was conducted with material testing system at a speed of 2mm/min. The experimental results were statistically analyzed using SPSS 17.0.

Results

(1) Significant differences of biomechanical properties occurred among normal BMD, osteoporotic and serious osteoporotic group (P<0.05). (2) Spearman analysis showed that BMD was positively correlated with the failure load and stiffness of lumbar vertebrae. (3) For each BMD group, significant differences of biomechanical properties were found between subgroup A and C, and between subgroup B and C (P<0.05). (4) For each BMD group, there was no statistical difference of biomechanical properties between subgroup A and B (P>0.05).

Conclusions

Entire endplate removal can significantly decrease the structural properties of lumbar vertebrae with little change in biomechanical properties by preservation of peripheral region of the endplate. BMD is positively correlated to the structural properties of the lumbar vertebrae.     

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.     

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.     

A method for estimation of lateral and vertical mobility of platycoelous vertebrae of tetrapods

A new method for the estimation of dorsoventral and lateral mobility of platycoelous vertebrae with V-shaped (radial) articular facets on the zygapophyses is developed. This vertebral pattern is observed in dinosaurs, some other fossil reptiles, and in the cervical and lumbar regions of mammals. Based on theoretical biomechanical analysis of the intervertebral discs and articulations between zygapophyses, the estimation formulas are developed and calibrated, using precise measurements of mobility between cervical vertebrae of domestic sheep. The method is applied to the presacral vertebrae of the horned dinosaur Protoceratops andrewsi. In its cervical, lumbar, and anterior thoracic regions, the differences between the calculated amplitudes of movements and the sought true values are expected to range within ±5°. As compared to the sheep, Protoceratops shows a greater lateral mobility in the presacral region and reduced vertical mobility in the cervical region.     

Functional implications of variation in lumbar vertebral count among hominins

As early as the 1970s, Robinson defined lumbar vertebrae according to their zygapophyseal orientation. He identified six lumbar elements in fossil Sts 14 Australopithecus africanus, one more than is commonly present in modern humans. It is now generally inferred that the modal number of lumbar vertebrae for australopiths and early Homo was six, from which the mode of five in later Homo is derived. The two central questions this study investigates are (1) to what extent do differences in human lumbar vertebral count affect lordotic shape and lumbar function, and (2) what does lumbar number variation imply about lumbar spine function in early hominins? To address these questions, I first outline a biomechanical model of lumbar number effect on lordotic function. I then identify relevant morphological differences in the human modal and extra-modal variants, which I use to test the model. These tests permit evaluation of the human L6 variant as a model for reconstructing early hominin modal number and spine function. Application of the biomechanical model in reconstructing australopith/early Homo lumbar spines highlights shared principles of Euler column strength and sagittal spine flexibility among early and modern hominins. Within modern humans, the extra-modal L6 variant has an extended series of three cranially positioned kyphotic vertebrae and strongly oblique zygapophyseal facets at the last lumbar level. Although they share the same radius and length of lumbar curvature, the L6 variant differs functionally from the L5 mode in its expanded range of sagittal flexion/extension and enhanced resistance to shear. Given the modal number of six lumbar vertebrae in australopiths and early Homo, lumbar spine mobility and strength would have been key properties of vertebral function in early bipeds whose upper and lower body segments were coupled by close approximation of the thorax and iliac crests.     

A videofluoroscopy-based tracking algorithm for quantifying the time course of human intervertebral displacements

The motions of individual intervertebral joints can affect spine motion, injury risk, deterioration, pain, treatment strategies, and clinical outcomes. Since standard kinematic methods do not provide precise time-course details about individual vertebrae and intervertebral motions, information that could be useful for scientific advancement and clinical assessment, we developed an iterative template matching algorithm to obtain this data from videofluoroscopy images. To assess the bias of our approach, vertebrae in an intact porcine spine were tracked and compared to the motions of high-contrast markers. To estimate precision under clinical conditions, motions of three human cervical spines were tracked independently ten times and vertebral and intervertebral motions associated with individual trials were compared to corresponding averages. Both tests produced errors in intervertebral angular and shear displacements no greater than 0.4° and 0.055 mm, respectively. When applied to two patient cases, aberrant intervertebral motions in the cervical spine were typically found to correlate with patient-specific anatomical features such as disc height loss and osteophytes. The case studies suggest that intervertebral kinematic time-course data could have value in clinical assessments, lead to broader understanding of how specific anatomical features influence joint motions, and in due course inform clinical treatments.     

Biomechanical implications of lumbar spinal ligament transection

Many lumbar spine surgeries either intentionally or inadvertently damage or transect spinal ligaments. The purpose of this work was to quantify the previously unknown biomechanical consequences of isolated spinal ligament transection on the remaining spinal ligaments (stress transfer), vertebrae (bone remodelling stimulus) and intervertebral discs (disc pressure) of the lumbar spine. A finite element model of the full lumbar spine was developed and validated against experimental data and tested in the primary modes of spinal motion in the intact condition. Once a ligament was removed, stress increased in the remaining spinal ligaments and changes occurred in vertebral strain energy, but disc pressure remained similar. All major biomechanical changes occurred at the same spinal level as the transected ligament, with minor changes at adjacent levels. This work demonstrates that iatrogenic damage to spinal ligaments disturbs the load sharing within the spinal ligament network and may induce significant clinically relevant changes in the spinal motion segment.     

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.     

Assessment of intervertebral disc degeneration-related properties using finite element models based on $$\uprho _H$$ -weighted MRI data

Biomechanics and Modeling in Mechanobiology - Quantitative magnetic resonance imaging (MRI) provides useful information about intervertebral disc (IVD) biomechanical properties, especially those in...