Effect of removing the nucleus pulposus on the deformation of the annulus fibrosus during compression of the intervertebral disc

Eighteen frozen ovine discs were bisected, in the mid-sagittal plane, to produce 36 specimens. The cut surfaces were marked at the inner and outer annulus boundaries of the annulus fibrosus, both anteriorly and posteriorly, with Alcian blue stain. The sections were sealed by a transparent plate, and thawed. A compression of 1mm at a rate of 0.2mms(-1) was applied. The displacements of the Alcian blue marks were measured from the video images, recorded during the tests, using interactive image analysis software. Before removal of the nucleus, the inner boundaries of the annulus moved outwards during compression (P<0.001, anterior; P=0.01, posterior). However, after removal of the nucleus, both inner boundaries moved inwards (P<0.001, anterior and posterior). The outer boundaries moved outwards both before and after removal of the nucleus (P<0.001). The results showed that total removal of the nucleus changes the response of the annulus to compression.     

Confined compression experiments on bovine nucleus pulposus and annulus fibrosus: sensitivity of the experiment in the determination of compressive modulus and hydraulic permeability

The biphasic material properties for nucleus pulposus tissue in confined compression have not been reported previously, and are required for a better understanding of intervertebral disc function and to provide material properties for use in finite-element models. The aims of this study were to determine linear and non-linear material properties for nucleus pulposus and annulus fibrosus tissues in confined compression, to define the influence of swelling conditions on these properties, and to determine the changes in the compressive modulus and hydraulic permeability induced by the repetition of the stress-relaxation experiment after a return to swelling pressure equilibrium. Specimens from caudal bovine nucleus and annulus were tested in confined compression stress-relaxation experiments and analyzed to quantify the compressive modulus and hydraulic permeability using linear and non-linear biphasic models. Our results suggested the use of confined swelling pre-test condition and non-linear biphasic model, which provided the material parameters with lowest relative variance and water content most representative of physiological conditions. Smaller compressive modulus and higher hydraulic permeability were obtained for the nucleus (H(A0)=0.31+/-0.04 MPa, k(0)=0.67+/-0.09 x 10(-15)m(4)/Ns) than for the annulus (H(A0)=0.74+/-0.13 MPa, k(0)=0.23+/-0.19 x 10(-15)m(4)/Ns), with relatively weak non-linearities. Strains up to 20% resulted in material properties that were significantly altered upon retesting. These altered material properties are an effort to quantify non-recoverable damage that occurs in disc tissue and suggest that in vivo exposure of disc tissues to low strain-rate and high-deformation loading conditions which outpace biological repair may result in altered mechanical behaviors.     

Penetrating annulus fibrosus injuries affect dynamic compressive behaviors of the intervertebral disc via altered fluid flow: an analytical interpretation

Extensive experimental work on the effects of penetrating annular injuries indicated that large injuries impact axial compressive properties of small animal intervertebral discs, yet there is some disagreement regarding the sensitivity of mechanical tests to small injury sizes. In order to understand the mechanism of injury size sensitivity, this study proposed a simple one dimensional model coupling elastic deformations in the annulus with fluid flow into and out of the nucleus through both porous boundaries and through a penetrating annular injury. The model was evaluated numerically in dynamic compression with parameters obtained by fitting the solution to experimental stress-relaxation data. The model predicted low sensitivity of mechanical changes to injury diameter at both small and large sizes (as measured by low and high ratios of injury diameter to annulus thickness), with a narrow range of high sensitivity in between. The size at which axial mechanics were most sensitive to injury size (i.e., critical injury size) increased with loading frequency. This study provides a quantitative hypothetical model of how penetrating annulus fibrosus injuries in discs with a gelatinous nucleus pulposus may alter disc mechanics by changing nucleus pulposus fluid pressurization through introduction of a new fluid transport pathway though the annulus. This model also explains how puncture-induced biomechanical changes depend on both injury size and test protocol.     

A comparison of uniaxial and biaxial mechanical properties of the annulus fibrosus: a porcine model

The annulus fibrosus of the intervertebral disk experiences multidirectional tension in vivo, yet the majority of mechanical property testing has been uniaxial. Therefore, our understanding of how this complex multilayered tissue responds to loading may be deficient. This study aimed to determine the mechanical properties of porcine annular samples under uniaxial and biaxial tensile loading. Two-layer annulus samples were isolated from porcine disks from four locations: anterior superficial, anterior deep, posterior superficial, and posterior deep. These tissues were then subjected to three deformation conditions each to a maximal stretch ratio of 1.23: uniaxial, constrained uniaxial, and biaxial. Uniaxial deformation was applied in the circumferential direction, while biaxial deformation was applied simultaneously in the circumferential and compressive directions. Constrained uniaxial consisted of a stretch ratio of 1.23 in the circumferential direction while holding the tissue stationary in the axial direction. The maximal stress and stress-stretch ratio (S-S) moduli determined from the biaxial tests were significantly higher than those observed during both the uniaxial tests (maximal stress, 97.1% higher during biaxial; p=0.002; S-S moduli, 117.9% higher during biaxial; p=0.0004) and the constrained uniaxial tests (maximal stress, 46.8% higher during biaxial; S-S moduli, 82.9% higher during biaxial). These findings suggest that the annulus is subjected to higher stresses in vivo when under multidirectional tension.     

Microstructural characterization of annulus fibrosus by ultrasonography: a feasibility study with an in vivo and in vitro approach

The main function of the intervertebral disc is biomechanical function, since it must resist repetitive high loadings, while giving the spine its flexibility and protecting the spinal cord from over-straining. It partially owes its mechanical characteristics to the lamellar architecture of its outer layer, the annulus fibrosus. Today, no non-invasive means exist to characterize annulus lamellar structure in vivo. The aim of this work was to test the feasibility of imaging annulus fibrosus microstructure in vivo with ultrasonography. Twenty-nine healthy adolescents were included. Ultrasonographies of L3–L4 disc were acquired with a frontal approach. Annulus fibrosus was segmented in the images to measure the thickness of the lamellae. To validate lamellar appearance in ultrasonographies, multimodality images of two cow tail discs were compared: ultrasonography, magnetic resonance and optical microscopy. In vivo average lamellar thickness was 229.7 ± 91.5 μm, and it correlated with patient body mass index and age. Lamellar appearance in the three imaging modalities in vitro was consistent. Lamellar measurement uncertainty was 7%, with good agreement between two operators. Feasibility of ultrasonography for the analysis of lumbar annulus fibrosus structure was confirmed. Further work should aim at validating measurement reliability, and to assess the relevance of the method to characterize annulus alterations, for instance in disc degeneration or scoliosis.

     

Targeting the annulus fibrosus of the intervertebral disc: <Emphasis Type="Italic">Col1a2-Cre(ER)T</Emphasis> mice show specific activity of Cre recombinase in the outer annulus fibrosus

Degeneration of the intervertebral disc (IVD) is a major underlying contributor to back pain—the single leading cause of disability worldwide. However, we possess a limited understanding of the etiology underlying IVD degeneration. To date, there are a limited number of mouse models that have been used to target proteins in specific compartments of the IVD to explore their functions in disc development, homeostasis and disease. Furthermore, the majority of reports exploring the composition and function of the outer encapsulating annulus fibrosus (AF) of the IVD have considered it as one tissue, without considering the numerous structural and functional differences existing between the inner and outer AF. In addition, no mouse models have yet been reported that enable specific targeting of genes within the outer AF. In the current report, we discuss these issues and demonstrate the localized activity of Cre recombinase in the IVD of Col1a2-Cre(ER)T;ROSA26mTmG mice possessing a tamoxifen-dependent Cre recombinase driven by a Cola2 promoter and distal enhancer and the mTmG fluorescent reporter. Following tamoxifen injection of 3-week-old Col1a2-Cre(ER)T;ROSA26mTmG mice, we show Cre activity specifically in the outer AF of the IVD, as indicated by expression of the GFP reporter. Thus, Col1a2-Cre(ER)T;ROSA26mTmG mice may prove to be a valuable tool in delineating the function of proteins in this unique compartment of the IVD, and in further exploring the compositional differences between the inner and outer AF in disc homeostasis, aging and disease.     

The recruitment of different compartments within a muscle depends on the mechanics of the movement

Muscles are commonly assumed to have uniform activations across their bellies. Yet animal studies have shown that different regions across a muscle can vary in their architecture, fibre type, levels of activation and the transfer of forces to the bones and tendons. The purpose of this study was to test whether regional variations in activity occurred across the soleus, medial gastrocnemius and lateral gastrocnemius in man, and whether these regional variations changed with altered mechanical demands on the limb. Arrays of surface electrodes were placed over these muscles and the electromyograms (EMG) measured for a range of cycling tasks where the resistance and pedal velocity was independently altered. Significant variations in the magnitude and timing of the EMG occurred across these muscles, which were most pronounced in the gastrocnemii and occurred in both the proximodistal and mediolateral directions. The patterns of variation across the muscles changed in response to the altered mechanical demands during the cycling. It is likely that the muscle fascicles in the gastrocnemii contribute varied mechanical functions to the contractions that depend on both their location within the muscle belly and on the mechanical requirements of the movement.     

Speciation within genomic networks: a case study based on Steatocranus cichlids of the lower Congo rapids

Hybridization in animals is a much more common phenomenon as previously thought and may have profound implications for speciation research. The cichlid genus Steatocranus (Teleostei: Cichlidae), a close relative to members of the East African cichlid radiations, radiated under riverine conditions in the lower Congo rapids and produced a small species flock. Previous phylogenetic analyses suggested that hybridization occurred and contributed to speciation in this genus. A re-analysis of an already published 2000 loci-AFLP data set explicitly testing for patterns of ancient gene flow provided strong evidence for a highly reticulate phylogenetic history of the genus. We provide, to our knowledge, the first example of a complex reticulate network in vertebrates, including multiple closely related species connected through ancient as well as recent gene flow. In this context, the limited validity of strictly bifurcating tree hypotheses as a phylogenetic basis for hypothesis testing in evolutionary biology is discussed.     

Population size estimates based on the frequency of genetically assigned parent–offspring pairs within a subsample

Estimating population density as precise as possible is a key premise for managing wild animal species. This can be a challenging task if the species in question is elusive or, due to high quantities, hard to count. We present a new, mathematically derived estimator for population size, where the estimation is based solely on the frequency of genetically assigned parent–offspring pairs within a subsample of an ungulate population. By use of molecular markers like microsatellites, the number of these parent–offspring pairs can be determined. The study's aim was to clarify whether a classical capture–mark–recapture (CMR) method can be adapted or extended by this genetic element to a genetic‐based capture–mark–recapture (g‐CMR). We numerically validate the presented estimator (and corresponding variance estimates) and provide the R‐code for the computation of estimates of population size including confidence intervals. The presented method provides a new framework to precisely estimate population size based on the genetic analysis of a one‐time subsample. This is especially of value where traditional CMR methods or other DNA‐based (fecal or hair) capture–recapture methods fail or are too difficult to apply. The DNA source used is basically irrelevant, but in the present case the sampling of an annual hunting bag is to serve as data basis. In addition to the high quality of muscle tissue samples, hunting bags provide additional and essential information for wildlife management practices, such as age, weight, or sex. In cases where a g‐CMR method is ecologically and hunting‐wise appropriate, it enables a wide applicability, also through its species‐independent use.     

Colonial differentiation in Streptomyces coelicolor depends on translation of a specific codon within the adpA gene

We identified adpA as an araC-like regulatory gene needed for colonial morphogenesis in Streptomyces coelicolor and showed that its activity depended on a unique TTA triplet corresponding to the leucyl-tRNA gene (bldA). These findings partially explained the dependence of aerial mycelium formation on a rare tRNA that is postulated to have developmental control functions.     

Effects of active site mutations in haemoglobin I from Lucina pectinata: a molecular dynamic study

Haemoglobin I from Lucina pectinata is a monomeric protein consisting of 142 amino acids. Its active site contains a peculiar arrangement of phenylalanine residues (PheB10, PheCD1 and PheE11) and a distal Gln at position E7. Active site mutations at positions B10, E7 and E11 were performed in deoxy haemoglobin I (HbI), followed by 10 ns molecular dynamic simulations. The results showed that the mutations induced changes in domains far from the active site producing more flexible structures than the native HbI. Distance analyses revealed that the heme pocket amino acids at positions E7 and B10 are extremely sensitive to any heme pocket residue mutation. The high flexibility observed by the E7 position suggests an important role in the ligand binding kinetics in ferrous HbI, while both positions play a major role in the ligand stabilisation processes. Furthermore, our results showed that E11Phe plays a pivotal role in protein stability.     

Delivering the goods: Activity-induced exocytosis and AMPA receptor insertion within a post-synaptic membrane compartment depends on syntaxin 4

The activity-dependent strengthening of neural transmission at individual synapses has long been postulated to underlie learning and memory in the brain, and current wisdom strongly suggests that molecular modifications within both the pre- and post-synaptic nerve terminals contribute to this strengthening process (i.e. long-term potentiation or LTP). At excitatory, glutamatergic synapses, the dynamic insertion and retrieval of ionotropic glutamate receptors into and from the post-synaptic plasma membrane have been implicated in synaptic plasticity, however, the site(s) for these trafficking events and the molecules involved have not be clearly elucidated. Biochemical studies have identified SNARE proteins as critical mediators of membrane fusion events in many cell types, including neurons, and several bacterial toxins are known to interfere with neurotransmission by disrupting the function of membrane-bound SNARE proteins, such as VAMP and syntaxin. Using high resolution imaging techniques, the authors have characterized the molecular processes underlying activity-driven membrane fusion events within dendritic spines, tiny membrane protrusions only 1-2 microns in size. Their data demonstrate that syntaxin 4 functions as a key SNARE protein for the exocytic insertion of glutamate receptors and the membrane trafficking events contributing to synaptic plasticity.     

Effect of pulse frequency on the osteogenic differentiation of mesenchymal stem cells in a pulsatile perfusion bioreactor

Perfusion bioreactors are a promising in vitro strategy to engineer bone tissue because they supply needed oxygen and nutrients and apply an osteoinductive mechanical stimulus to osteoblasts within large porous three-dimensional scaffolds. Model two-dimensional studies have shown that dynamic flow conditions (e.g., pulsatile oscillatory waveforms) elicit an enhanced mechanotransductive response and elevated expression of osteoblastic proteins relative to steady flow. However, dynamic perfusion of three-dimensional scaffolds has been primarily examined in short term cultures to probe for early markers of mechanotransduction. Therefore, the objective of this study was to investigate the effect of extended dynamic perfusion culture on osteoblastic differentiation of primary mesenchymal stem cells (MSCs). To accomplish this, rat bone marrow-derived MSCs were seeded into porous foam scaffolds and cultured for 15 days in osteogenic medium under pulsatile regimens of 0.083, 0.050, and 0.017 Hz. Concurrently, MSCs seeded in scaffolds were also maintained under static conditions or cultured under steady perfusion. Analysis of the cells after 15 days of culture indicated that alkaline phosphatase (ALP) activity, mRNA expression of osteopontin (OPN), and accumulation of OPN and prostaglandin E(2) were enhanced for all four perfusion conditions relative to static culture. ALP activity, OPN and OC mRNA, and OPN protein accumulation were slightly higher for the intermediate frequency (0.05 Hz) as compared with the other flow conditions, but the differences were not statistically significant. Nevertheless, these results demonstrate that dynamic perfusion of MSCs may be a useful strategy for stimulating osteoblastic differentiation in vitro.     

Biologically based insecticides for the control of immature Australian mosquitoes: a review

Abstract  Our review outlines changes in insecticide usage for mosquito control against Australian mosquitoes, with a focus on biologically based insecticides. The most widely used microbial insecticide is Bacillus thuringiensis var. israelensis de Barjac ( Bti ) and it is regularly used in estuaries and freshwater. In comparison, the use of Bacillus sphaericus (Neide) is limited to freshwater. The only insect growth regulator registered for mosquito control in Australia is s -methoprene, and it is used in both estuaries and freshwater. Although biologically based insecticides have been used for more than 10 years, there remains knowledge gaps surrounding the use of specific formulations in specific habitats: for example, VectoBac WG ( Bti ) and VectoBac G ( Bti ) in containers containing freshwater, or Altosid pellets ( s -methoprene) in ground-pool freshwater habitats. Where broad-scale mosquito control programs have been implemented in Australia, a reduction in the incidence of mosquito-borne disease, mainly Ross River virus, has been recorded. Therefore, the future application of these products is supported. The use of insecticides should be integrated with public education, biological control, physical habitat modification and coupled with spatial risk assessment.     

A study of the Coriolis effect on the fluid flow profile in a centrifugal bioreactor

Increasing demand for tissues, proteins, and antibodies derived from cell culture is necessitating the development and implementation of high cell density bioreactors. A system for studying high density culture is the centrifugal bioreactor (CCBR), which retains cells by increasing settling velocities through system rotation, thereby eliminating diffusional limitations associated with mechanical cell retention devices. This article focuses on the fluid mechanics of the CCBR system by considering Coriolis effects. Such considerations for centrifugal bioprocessing have heretofore been ignored; therefore, a simpler analysis of an empty chamber will be performed. Comparisons are made between numerical simulations and bromophenol blue dye injection experiments. For the non‐rotating bioreactor with an inlet velocity of 4.3 cm/s, both the numerical and experimental results show the formation of a teardrop shaped plume of dye following streamlines through the reactor. However, as the reactor is rotated, the simulation predicts the development of vortices and a flow profile dominated by Coriolis forces resulting in the majority of flow up the leading wall of the reactor as dye initially enters the chamber, results are confirmed by experimental observations. As the reactor continues to fill with dye, the simulation predicts dye movement up both walls while experimental observations show the reactor fills with dye from the exit to the inlet. Differences between the simulation and experimental observations can be explained by excessive diffusion required for simulation convergence, and a slight density difference between dyed and un‐dyed solutions. Implications of the results on practical bioreactor use are also discussed. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effect of surfactant concentration, compression ratio and compression rate on the surface activity and dynamic properties of a lung surfactant

This paper reports dynamic surface tension experiments of a lung surfactant preparation, BLES, for a wide range of concentrations, compression ratios and compression rates. These experiments were performed using Axisymmetric Drop Shape Analysis-Constrained Sessile Drop (ADSA-CSD). The main purpose of the paper is to interpret the results in terms of physical parameters using the recently developed Compression-Relaxation Model (CRM). In the past, only the minimum surface tension was used generally for the characterization of lung surfactant films; however, this minimum value is not a physical parameter and depends on the compression protocol. CRM is based on the assumption that the dynamic surface tension response is governed by surface elasticities, adsorption and desorption of components of the lung surfactant. The ability of CRM to fit the surface tension response closely for a wide variety of parameters (compression ratio, compression rate and surfactant concentration) and produce sensible values for the elastic and kinetic parameters supports the validity of CRM.     

Interchange of catalytic activity within the 2-enoyl-coenzyme A hydratase/isomerase superfamily based on a common active site template.

The structures and chemical pathways associated with the members of the 2-enoyl-CoA hydratase/isomerase enzyme superfamily are compared to show that a common active site design provides the members of this family with a CoA binding site, an expandable acyl binding pocket, an oxyanion hole for binding/polarizing the thioester C=O, and multiple active site stations for the positioning of acidic and basic amino acid side chains for use in proton shuttling. It is hypothesized that this active site template can be tailored to catalyze a wide range of chemical transformations through strategic positioning of acid/base residues among the active site stations. To test this hypothesis, the active site of one member of the 2-enoyl-CoA hydratase/isomerase family, 4-chlorobenzoyl-CoA dehalogenase, was altered by site-directed mutagenesis to include the two glutamate residues functioning in acid/base catalysis in a second family member, crotonase. Catalysis of the syn hydration of crotonyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoA dehalogenase, was shown to occur with the structurally modified 4-chlorobenzoyl-CoA dehalogenase at kcat = 0.06 s-1 and Km = 50 microM.     

Shoulder muscle function depends on elbow joint position: an illustration of dynamic coupling in the upper limb

Shoulder muscle function has been documented based on muscle moment arms, lines of action and muscle contributions to contact force at the glenohumeral joint. At present, however, the contributions of individual muscles to shoulder joint motion have not been investigated, and the effects of shoulder and elbow joint position on shoulder muscle function are not well understood. The aims of this study were to compute the contributions of individual muscles to motion of the glenohumeral joint during abduction, and to examine the effect of elbow flexion on shoulder muscle function. A three-dimensional musculoskeletal model of the upper limb was used to determine the contributions of 18 major muscles and muscle sub-regions of the shoulder to glenohumeral joint motion during abduction. Muscle function was found to depend strongly on both shoulder and elbow joint positions. When the elbow was extended, the middle and anterior deltoid and supraspinatus were the greatest contributors to angular acceleration of the shoulder in abduction. In contrast, when the elbow was flexed at 90°, the anterior deltoid and subscapularis were the greatest contributors to joint angular acceleration in abduction. This dependence of shoulder muscle function on elbow joint position is explained by the existence of dynamic coupling in multi-joint musculoskeletal systems. The extent to which dynamic coupling affects shoulder muscle function, and therefore movement control, is determined by the structure of the inverse mass matrix, which depends on the configuration of the joints. The data provided may assist in the diagnosis of abnormal shoulder function, for example, due to muscle paralysis or in the case of full-thickness rotator cuff tears.     

Membrane bioreactor for pharmaceutically active compounds removal: Effects of carbamazepine on mixed microbial communities implied in the treatment

The aim of this work consists in evaluating the influence of carmabazepine (CBZ) (i) on the endogenous and exogenous respirations, in batch reactors, of bacterial populations taken from a conventional activated sludge process (CAS) and a pilot-scale membrane bioreactor (MBR) and (ii) on COD removal, sludge production and oxygen requirement of a lab-sale MBR system. In batch experiments, the increase in endogenous oxygen needs suggests an increase in maintenance requirements, essentially to manage the chemical stress induced by the CBZ presence. The decrease of exogenous oxygen needs seems to suggest a change in the metabolic pathways of the substrate or a change in the active bacterial species. However, in spite of these momentary changes, no inhibition is observed in the presence of CBZ in the test tank. This result is confirmed by the MBR experiment where no significant difference COD removal, sludge production and oxygen requirement is observed, with and without CBZ.     

基于灰色拓扑理论的草地生态系统损害基线动态预测

生态环境损害鉴定评估是生态环境损害赔偿制度有效执行的关键。生态环境损害基线贯穿生态环境损害鉴定评估的整个过程,是生态环境损害确认与损害修复的重要依据与标准。历史数据法是损害基线确认的优先采用方法,以损害发生前3年平均值作为静态基线值,难以体现基线的动态性与不确定性,因此生态环境损害基线的动态研究尤为重要。选取净初级生产力(NPP)作为草地生态系统损害表征指标,以锡林浩特市巴彦宝力格矿区未开发建设前2000—2008年NPP历史数据为基础,运用灰色拓扑模型对矿区2009—2011年年均NPP基线值进行动态预测,并将其与静态基线值进行比较,探讨该方法的可行性。结果表明:灰色拓扑模型建模精度高,相对误差小,该模型预测平均相对误差为2.88%,且2009—2011年的拟合曲线与实际变化曲线相一致,将该模型预测结果作为动态基线值比静态基线值更能反映现实情况。构建的植被NPP基线动态预测方法为基线的确定提供一种解决思路,具有一定的应用价值与科学合理性。