Multi-scale Structural and Tensile Mechanical Response of Annulus Fibrosus to Osmotic Loading

This study investigates differential multi-scale structure and function relationships of the outer and inner annulus fibrosus (AF) to osmotic swelling in different buffer solutions by quantifying tensile mechanics, glycoasamino-glycan(GAG) content, water content and tissue swelling, and collagen fibril ultrastructure. In the outer AF, the tensile modulus decreased by over 70% with 0.15 M PBS treatment but was unchanged with 2 M PBS treatment. Moreover, the modulus loss following 0.15 M PBS treatment was reversed when followed by 2 M PBS treatment, potentially from increased interfibrillar and interlamellar shearing associated with fibril swelling. In contrast, the inner AF tensile modulus was unchanged by 0.15 M PBS treatment and increased following 2 M treatment. Transmission electron microscopy revealed that the mean collagen fibril diameters of the untreated outer and inner AF were 87.8 ± 27.9 and 71.0 ± 26.9 nm, respectively. In the outer AF, collagen fibril swelling was observed with both 0.15 M and 2 M PBS treatments, but inherently low GAG content remained unchanged. In the inner AF, 2 M PBS treatment caused fibril swelling and GAG loss, suggesting that GAG plays a role in maintaining the structure of collagen fibrils leading to modulation of the native tissue mechanical properties. These results demonstrate important regional variations in structure and composition, and their influence on the heterogeneous mechanics of the AF. Moreover, because the composition and structure is altered as a consequence of progressive disk degeneration, quantification of these interactions is critical for study of the AF pathogenesis of degeneration and tissue engineering     

A Nonlinear Hyperelastic Mixture Theory Model for Anisotropy, Transport, and Swelling of Annulus Fibrosus

A precise knowledge of the local mechanical and chemical environment around the nerve endings and disc cells in the annulus fibrosus will shed insight on understanding the mechanism of low-back pain and disc degeneration. It would also present an effective tool for the studies of the intervertebral disc structure-function relationship and provide guidance to disc tissue engineering. Experimental difficulties preclude the direct and simultaneous measurement of many of the important physical quantities, such as annulus pressurization, nutrient and electrolyte transport, and mechanical and swelling deformation. Considering that many of these quantities are coupled and that the annulus is highly anisotropic, interpretation of the results would be extremely challenging without an appropriate theoretical framework. In this study, we develop a nonlinear hyperelastic fiber-reinforced continuum mixture theory model for the annulus fibrosus. Special attention is given to the anisotropic nature of the annulus. On the basis of the lamella structure of annulus, and derived from a Helmholtz energy function, a locally transversely isotropic stress-strain relation is adopted for explicit representation of the collagen fiber orientations in general finite deformation situation. The exponential form of the Helmholtz energy function naturally reduces to the infinitesimal deformation form, and the equivalence between the current model coefficients and engineering elastic constants is established under the infinitesimal deformation. This model is able to describe the anisotropic finite and infinitesimal deformation, tension-compression nonlinearity, osmotic swelling, pressurization, electrical potential and current, and water and ion transports as well as the electroneutral nutrient (or growth factor) transport within the annulus.     

Structure and Composition of Proteoglycans From Human Annulus Fibrosus

Proteoglycans were extracted from ground human lumbar annuli fibrosi with 4M guanidinium chloride and purified by means of associative equilibrium density gradient centrifugation. The proteoglycan preparations contained chondroitin sulphate, keratan sulphate and hyaluronic acid, but no dermatan sulphate. Degradation experiments suggested that the proteoglycans contain three regions: a chondroitin sulphate-rich region, a keratan sulphate-rich region and a region that binds to hyaluronic acid, thus allowing proteoglycan aggregates to be formed. The keratan sulphate-rich region seemed to be more prominent than in bovine hyaline cartilage proteoglycans. The model for the structure of bovine hyaline cartilage proteoglycans, Hascall and Heinegård,¹ seems to be applicable to the proteoglycans from human annulus fibrosus. The amino acid composition of annulus fibrosus proteoglycans is very similar to that of bovine hyaline cartilage proteoglycans.     

Elastic Fibers Enhance the Mechanical Integrity of the Human Lumbar Anulus Fibrosus in the Radial Direction

The anulus fibrosus of the human lumbar intervertebral disc has a complex, hierarchical structure comprised of collagens, proteoglycans, and elastic fibers. Recent histological studies have suggested that the elastic fiber network may play an important functional role. In this study, it was hypothesized that elastic fibers enhance the mechanical integrity of the extracellular matrix in the radial orientation, perpendicular to the plane containing the collagen fibers. Using a combination of biochemically verified enzymatic treatments and biomechanical tests, it was demonstrated that degradation of elastic fibers resulted in a significant reduction in both the initial modulus and the ultimate modulus, and a significant increase in the extensibility, of radially oriented anulus fibrosus specimens. Separate treatments and mechanical tests were used to account for any changes attributable to non-specific degradation of glycosaminoglycans. Additionally, histological assessments provided a unique perspective on structural changes in the elastic fiber network in radially oriented specimens subjected to tensile deformations. The results of this study demonstrate that elastic fibers play an important and unique role in the mechanical properties of the anulus fibrosus, and provide the basis for the development of improved material models to describe intervertebral disc mechanical behavior.     

Biaxial Testing of Human Annulus Fibrosus and Its Implications for a Constitutive Formulation

Internal pressure in the healthy human annulus fibrosus leads to multiaxial stress in vivo, yet uniaxial tests have been used exclusively to characterize its in vitro mechanical response and to determine its elastic strain energy function (W). We expected that biaxial tension tests would provide unique and necessary data for characterizing the annular material response, and thereby, for determining W. We performed uniaxial and biaxial tests on specimens of annulus, then developed an objective methodology for defining an appropriate form for W that considers data from multiple experiments simultaneously and allows the data to dictate more directly the form and the number of parameters needed. We found that the stresses attained in the biaxial tests were higher, while the strains were considerably lower, than those observed in the uniaxial tests. A comparison of strain energy functions determined from the different data sets demonstrated that constitutive models derived from uniaxial data could not predict annulus behavior in biaxial tension and vice versa. Since the annulus is in a state of multaxial stress in vivo, we conclude that uniaxial tests alone are insufficient to prescribe a physiologically relevant W for this tissue.     

Anisotropic Diffusive Transport in Annulus Fibrosus: Experimental Determination of the Diffusion Tensor by FRAP Technique

The annulus fibrosus (AF) of the intervertebral disc (IVD) exhibits a fiber-organized structure which is responsible for anisotropic and inhomogeneous mechanical and transport properties. Due to its particular morphology, nutrient transport within AF is regulated by complex transport kinetics. This work investigates the diffusive transport of a small solute in the posterior and anterior regions of AF since diffusion is the major transport mechanism for low molecular weight nutrients (e.g., oxygen and glucose) in IVD. Diffusion coefficient (D) of fluorescein (332 Da) in bovine coccygeal AF was measured in the three major (axial, circumferential, and radial) directions of the IVD by means of fluorescence recovery after photobleaching (FRAP) technique. It was found that the diffusion coefficient was anisotropic and inhomogeneous. In both anterior and posterior regions, the diffusion coefficient in the radial direction was found to be the lowest. Circumferential and axial diffusion coefficients were not significantly different in both posterior and anterior regions and their values were about 130% and 150% the value of the radial diffusion coefficient, respectively. The values of diffusion coefficients in the anterior region were in general higher than those of corresponding diffusion coefficients in the posterior region. This study represents the first quantitative analysis of anisotropic diffusion transport in AF by means of FRAP technique and provides additional knowledge on understanding the pathways of nutritional supply into IVD.     

Leptin Induces Terminal Differentiation of Rat Annulus Fibrosus Cells via Activation of MAPK Signaling

Both leptin and its receptor are expressed in rat annulus fibrosus (AF) cells. However, little is known about their role and mechanism during disc degeneration. The mitogen activating protein kinase (MAPK) pathway which mediates leptin‐induced terminal differentiation of rat AF cells was analyzed using PCR, Western‐blot and immunocytochemistry. It was found that leptin‐induced AF cells terminal differentiation, which may be attributed to upregulated p38 and ERK1/2 phosphorylation, however, JNK phosphorylation was not observed. Specific inhibitors of p38 or ERK1/2, but not JNK, could inhibit the stimulative activity of leptin on collagen X and MMP‐13 protein levels. This study, for the first time, shows that the MAPK pathway, especially p38 and ERK1/2 signaling, plays a distinct role in leptin‐induced AF cells terminal differentiation. Anat Rec, 296:1806–1812, 2013. © 2013 Wiley Periodicals, Inc.     

腰4、5间盘纤维环结构形态的细微观察

观测成人腰椎间盘纤维环纤维层水平的细微解剖结构。用显微外科技术将腰间盘纤维环分离,每层纤维均分为8个观测点,测量纤维走向与水平面的夹角等数值。结果表明:各测点处的纤维走向角α,在水平面,沿径向由外向内,随层数的增加不断变大,最小纤维走向角发生在前正中处,其值为25°~30°;在同一层,纤维走向角由前向后不断变大,在椎间盘正后方为70°~90°,即此部分纤维走向与后纵韧带走向基本一致;用归一化方程,归一化直线可以非常方便地求出任一层、任一点处的纤维走向角α;腰椎间盘纤维环的前方、左右两侧皆有交织现象发生,左右两侧交织区域多些,仅后方正中附近未发现相临两层的交织现象。由此可见,腰椎间盘纤维环纤维具有特殊的细微结构,与其功能相适应。     

A Microstructurally Motivated Model of the Mechanical Behavior of Tissue Engineered Blood Vessels

Mechanical models have potential to guide the development and use of engineered blood vessels as well as other engineered tissues. This paper presents a microstructurally motivated, pseudoelastic, mechanical model of the biaxial mechanics of engineered vessels in the physiologic pressure range. The model incorporates experimentally measured densities and alignments of engineered collagen. Specifically, these microstructural and associated mechanical inputs were measured directly from engineered blood vessels that were cultured over periods of 5–7.5 weeks. To the best of our knowledge, this is the first successful application of either a phenomenological or a microstructurally motivated mechanical model to engineered vascular tissues. Model development revealed the need to use novel theoretical configurations to describe the strain history of engineered vessels. The constitutive equations developed herein suggested that collagen remodeled between 5 and 7.5 weeks during a 7.5-week culture period. This remodeling led to strain energies for collagen that differed with alignment, which likely resulted from undulations that varied with alignment. Finally, biaxial data emphasized that axial extensions increase stresses in engineered vessels in the physiologic pressure range, thereby providing a guideline for surgical use: engineered vessels should be implanted at appropriate axial extension to minimize adverse stress responses.     

腰椎4、5间盘纤维环纤维层力学性能的实验研究

测试成人腰椎间盘纤维环纤维层的力学性能。用显微外科技术将纤维环逐层分离,每层纤维平均分成8个测点,每层每测点处按纤维走向切取5个试件,长度l=15~20mm,宽b=1~1.5mm,厚t=0.1~0.5mm。进行拉伸实验测得应力-应变曲线,并给出拟合曲线及方程,得出弹性模量、破坏应变、破坏应力、测点处的力学性能参数和方程。测试结果表明:各测点处,沿径向,由外向内弹性模量皆随层数的增加,不断变小;每层纤维弹性模量由前侧向后侧逐渐变小;破坏应力由外层向内层线性递减;破坏应变外部9层随层数增加略有提高,9层以后基本上为一常数(0.34±0.14)。可见,腰椎间盘纤维环纤维具有特殊的力学性能,与其功能相适应,与腰椎疾病的发生有密切的关系。     

Worldview and Health Promoting Behavior: A Causal Model

The present study investigated the manner in which Pepper's (1942) worldview theory relates to health promoting behavior. A sample of 259 subjects completed a battery of inventories measuring worldview, health promoting behavior (HPB), social class, and sex. The data were analyzed by means of structural equation modeling using the statistical program for the social sciences (SPSS) and the analysis of moment and structure (AMOS) computer programs. The results support the idea that a modest relationship exists between worldview and HPB, with organismic thinkers more likely than mechanistic thinkers to engage in HPB. There was also a slight indirect effect of sex on worldview and HPB, with women more likely to endorse an organismic worldview and therefore more likely to engage in HPB than men. No relationship was found between socioeconomic status and HPB.     

A Microstructural Hyperelastic Model of Pulmonary Arteries Under Normo- and Hypertensive Conditions

This work represents the first application of a statistical mechanics based microstructural orthotropic hyperelastic model to pulmonary artery mechanics under normotensive and hypertensive conditions. The model provides an analogy between the entangled network of long molecular chains and the structural protein framework seen in the medial layer, and relates the mechanical response at macro-level to the deformation (entropy change) of individual molecular chains at the micro-level. A finite element approach was adopted to implement the model. Material parameters were determined via comparing model output to measured pressure–stretch results from normotensive and hypertensive trunks and branches obtained from a rat model of pulmonary arterial hypertension. Results from this initial study show that this model appears reasonable for the study of hyperelastic and anisotropic pulmonary artery mechanics. Typical tangent modulus values ranged from 200 to 800 kPa for normotensive arteries—this increased to beyond 1 MPa for hypertensive vessels. Our study also provokes the hypothesis that increase of cross-linking density may be one mechanism by which the pulmonary artery stiffens in hypertension.     

恒流电刺激下神经纤维的非线性行为

生命系统中存在着丰富的非线性现象。我们对于恒流电刺激下的单根神经纤维兴奋性进行了计算机仿真,观察到了非线性振荡现象,得到了膜电位稳定态和周期态与刺激电流强度的关系,以及周期振荡频率与刺激强度间的关系。     

Mechanical Properties of Regenerated Bombyx mori Silk Fibers and Recombinant Silk Fibers Produced by Transgenic Silkworms

Regenerated silk fibroin fibers from the cocoons of silkworm, Bombyx mori, were prepared with hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or hexafluoroacetone-trihydrate (HFA), as dope solvents and methanol as coagulation solvent. The regenerated fiber prepared from the HFIP solution showed slightly larger tensile strength when the draw ratio is 1:3 than that of native silk fiber, but the strength of the regenerated fiber with draw ratio 1:3 from the HFA solution is much lower than that of native silk fiber. This difference in the tensile strength of the regenerated silk fibers between two dope solvents comes from the difference in the long-range orientation of the crystalline region rather than that of short-range structural environment such as the fraction of β-sheet structure. The increase in the biodegradation was observed for the regenerated silk fiber compared with native silk fiber. Preparations of regenerated silk fibroin fibers containing spider silk sequences were obtained by mixing silk fibroins and silk-like proteins with characteristic sequences from a spider, Naphila clavipes, to produce drag-line silk in E. coli in the fluoro solvents. A small increase in the tensile strength was obtained by adding 5% (w/w) of the silk-like protein to the silk fibroin. The production of silk fibroin fibers with these spider silk sequences was also performed with transgenic silkworms. Small increase in the tensile strength of the fibers was obtained without significant change in the elongation-at-break.     

Contractility Affects Stress Fiber Remodeling and Reorientation of Endothelial Cells Subjected to Cyclic Mechanical Stretching

We studied the effect of contractility on stress fiber remodeling and orientation response of human aortic endothelial cells subjected to cyclic mechanical stretching. The cells were grown on silicone membranes subjected to 10% cyclic pure uniaxial stretching in the presence or absence of 2,3 butanedione monoxime (BDM), a proven inhibitor of cellular contractility. It was found that treatment of the cells with BDM (40 mM) abolished stress fibers and blocked cell reorientation in response to cyclic stretching, indicating that contractility is required for these two cellular responses. When cells were stretched in the presence of N-acetylcysteine (NAC, 20 mM), a hydrogen peroxide (H₂O₂) scavenger, stress fibers were still formed and the cells reoriented—but more slowly. Specifically, compared with untreated cells, NAC treated cells after 0.5, 1, and 3 h of 10% stretching had significantly (p < 0.005) less skewed orientation distributions than those of untreated cells. After the cells were treated with both NAC (20 mM) and nordihydroguaiaretic acid (NDGA, 50 M), another antioxidant, however, stress fibers were abolished and cell reorientation was completely blocked. These results indicate that reactive oxygen species (ROS), including (H₂O₂) affect stress fiber remodeling and reorientation of endothelial cells in response to cyclic stretching. We suggest that the effect of ROS on stress fiber remodeling and cell reorientation is due to the ability of ROS to regulate cellular contractility, which is crucial for these cellular responses. © 2000 Biomedical Engineering Society. PAC00: 8717-d, 8719Rr, 8715La     

聚D,L-乳酸/羟基磷灰石复合纤维的力学性能研究

采用熔融纺丝法制备PDLLA／HA复合纤维，探讨PDLLA／HA复合纤维的力学性能及影响因素和性能变化规律。实验结果表明：在分子量为12万的PDLLA中，加入一定量4～20μm的HA颗粒能提高复合纤维的力学性能。在PDLLA基体中添加HA的质量分数以10％为宜，以此配比制备的复合纤维的断裂强度高于其它配比复合纤维的断裂强度。采用分子量为20～30万的PDLLA制备的复合纤维断裂强度高，性能优异。复合纤维断裂强度随纤维直径的增加而下降，在直径为40～60μm时，复合纤维断裂伸长率高，弹性好。     

FTIR‐Spectroscopy on Segmental Reorientation of a Nematic Elastomer under External Mechanical Fields

Summary: Fourier transform infrared (FTIR) spectroscopy with polarized light is employed to study the segmental orientation and the order of a nematic liquid crystalline elastomers (NLCEs) with a monodomain structure in response to an external mechanical field. The reorientation and the order parameter of the different molecular moieties are analyzed in detail, revealing information about angular excursion in the rearrangement of the mesogens, the spacer molecules, and the main‐chain. In case of an elongation of the NLCE films perpendicular to the initial mesogen orientation, no reorientation or change of order is observed for an elongation ratio less than 1.3. At higher strain, a molecular reorientation process is induced on all molecular segments and the order of alignment is decreased. When the NLCE‐films are stretched parallel to the mesogens, no molecular reorientation takes place and the order parameters show no significant change.

Angular reorientation of different molecular moieties at mechanical strain perpendicular to the initial mesogen orientation.     

A Smectic A Liquid Single Crystal Elastomer (LSCE): Phase Behavior and Mechanical Anisotropy

The analysis of the phase behavior of a smectic A (S_A) elastomer reveals a nematic phase existing within a small temperature range below the isotropic state. Stress‐optical measurements in the pretransformational regime of the isotropic state indicate smectic as well as nematic fluctuations yielding a critical exponent of γ = 0.65. The formation of the liquid single crystal elastomer (LSCE) at the isotropic to liquid crystalline phase transformation equals a nematic LSCE. At the nematic to S_A phase transformation, the orientation of the director remains constant while the tendency of the network strands towards an oblate equilibrium conformation is suppressed by the high modulus parallel to the smectic layer normal. The mechanical anisotropy of the S_A‐LSCE as a function of the temperature is characterized by entropy elasticity perpendicular to the smectic layer normal. Parallel to the layer normal the mechanical response is determined by the enthalpy elastic response of the smectic layers having a modulus larger by about two orders of magnitude. In this direction the modulus decreases linearly with increasing temperature and reflects the falling stability of the layers. Accordingly, above a deformation of about 2% the homogeneous layered structure breaks down at a threshold stress that also falls linearly with increasing temperature while the threshold strain remains constant at about 2% elongation.