Material properties of the human lumbar facet joint capsule

The human facet joint capsule is one of the structures in the lumbar spine that constrains motions of vertebrae during global spine loading (e.g., physiological flexion). Computational models of the spine have not been able to include accurate nonlinear and viscoelastic material properties, as they have not previously been measured. Capsules were tested using a uniaxial ramp-hold protocol or a haversine displacement protocol using a commercially available materials testing device. Plane strain was measured optically. Capsules were tested both parallel and perpendicular to the dominant orientation of the collagen fibers in the capsules. Viscoelastic material properties were determined. Parallel to the dominant orientation of the collagen fibers, the complex modulus of elasticity was E*=1.63MPa, with a storage modulus of E'=1.25MPa and a loss modulus of: E" =0.39MPa. The mean stress relaxation rates for static and dynamic loading were best fit with first-order polynomials: B(epsilon) = 0.1110epsilon-0.0733 and B(epsilon)= -0.1249epsilon + 0.0190, respectively. Perpendicular to the collagen fiber orientation, the viscous and elastic secant moduli were 1.81 and 1.00 MPa, respectively. The mean stress relaxation rate for static loading was best fit with a first-order polynomial: B (epsilon) = -0.04epsilon - 0.06. Capsule strength parallel and perpendicular to collagen fiber orientation was 1.90 and 0.95 MPa, respectively, and extensibility was 0.65 and 0.60, respectively. Poisson's ratio parallel and perpendicular to fiber orientation was 0.299 and 0.488, respectively. The elasticity moduli were nonlinear and anisotropic, and capsule strength was larger aligned parallel to the collagen fibers. The phase lag between stress and strain increased with haversine frequency, but the storage modulus remained large relative to the complex modulus. The stress relaxation rate was strain dependent parallel to the collagen fibers, but was strain independent perpendicularly.     

On the anisotropy of the canine diaphragmatic central tendon

We studied the mechanical and anatomical anisotropy of the canine diaphragmatic central tendon (CT). Dumb-bell-shaped strips with effective dimensions of 10 x 2 mm (length x width) were cut from different regions of the canine diaphragmatic CT in two different orientations relative to the direction of neighboring muscle fibers. Specimens sampled with their long axial dimension oriented parallel to the neighboring muscle fibers were named Group-1 and those sampled with an orientation perpendicular to the neighboring muscle fibers were named Group-2. Results from one-dimensional stress-strain and tensile failure strength tests revealed that the CT is a nonlinear, inelastic, and anisotropic material. Group-1 specimens were found to have a higher stiffness, higher failure strength and higher strain energy density at failure than Group-2 specimens. Polarized microscopy showed that multiple sheets of collagen fiber bundles formed an orthogonal network in the tendon. Collagen fiber bundles along Group-1 direction formed parallel trajectory lines connecting the neighboring costal and crural muscles; bundles along Group-2 direction were observed to orient 90 degrees away. At the central apex region of the CT, collagen bundles of Group-1 formed a fan-like trajectory pattern. This collagen network architecture was compared favorably to the trajectories of an approximated principal stress field in the CT due to simulated contractile forces from its adjacent costal and crural muscles. These combined results suggest a structure-function relationship for the anatomical and mechanical anisotropy in the canine diaphragmatic CT.     

Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering

Many musculoskeletal tissues exhibit significant anisotropic mechanical properties reflective of a highly oriented underlying extracellular matrix. For tissue engineering, recreating this organization of the native tissue remains a challenge. To address this issue, this study explored the fabrication of biodegradable nanofibrous scaffolds composed of aligned fibers via electrospinning onto a rotating target, and characterized their mechanical anisotropy as a function of the production parameters. The characterization showed that nanofiber organization was dependent on the rotation speed of the target; randomly oriented fibers (33% fiber alignment) were produced on a stationary shaft, whereas highly oriented fibers (94% fiber alignment) were produced when rotation speed was increased to 9.3m/s. Non-aligned scaffolds had an isotropic tensile modulus of 2.1+/-0.4MPa, compared to highly anisotropic scaffolds whose modulus was 11.6+/-3.1MPa in the presumed fiber direction, suggesting that fiber alignment has a profound effect on the mechanical properties of scaffolds. Mechanical anisotropy was most pronounced at higher rotation speeds, with a greater than 33-fold enhancement of the Young's modulus in the fiber direction compared to perpendicular to the fiber direction when the rotation speed reached 8m/s. In cell culture, both the organization of actin filaments of human mesenchymal stem cells and the cellular alignment of meniscal fibroblasts were dictated by the prevailing nanofiber orientation. This study demonstrates that controllable and anisotropic mechanical properties of nanofibrous scaffolds can be achieved by dictating nanofiber organization through intelligent scaffold design.     

Characterization of collagen fiber architecture in the canine diaphragmatic central tendon.

The diaphragmatic central tendon (DCT), a collagenous soft tissue membrane, acts as a mechanical buffer between the costal and crural muscles. Its direction of mechanical anisotropy has been shown to correspond to the collagen fiber preferred directions. These preferred directions were determined by gross histological examination, and were thus qualitative. In this work we quantified the collagen fiber architecture throughout the DCT using small angle light scattering (SALS). Helium-Neon laser light was passed through tendon specimens and the resultant scattered light distribution, which characterized the local collagen fiber architecture, was recorded with a linear array of five photodiodes. Throughout the DCT two distinct collagen fiber populations were consistently found. For each population three parameters were determined: 1) the preferred directions of collagen fibers, 2) the volume fraction (Vf) of fibers, 3) OI, an orientation index, which ranges from 0 percent for a random network to 100 percent for a perfectly oriented network. Vector maps were used to display results from 1) and 2), and showed a primary group (G1) going from the crural to costal muscles and a secondary one (G2) running perpendicular to G1. Comparisons of Vf between G1 and G2 showed that G1 contained about three times as many fibers as G2, a ratio similar to that found for the degree of mechanical anisotropy. OI were found to be about 60 percent, indicating a high degree of orientation, with no significant regional or population differences (p less than 0.05). These quantitative results suggest that throughout the DCT the degree of mechanical anisotropy is controlled exclusively by Vf.     

Regulation of stretch-induced JNK activation by stress fiber orientation

Cyclic mechanical stretch associated with pulsatile blood pressure can modulate cytoskeletal remodeling and intracellular signaling in vascular endothelial cells. The aim of this study was to evaluate the role of stretch-induced actin stress fiber orientation in intracellular signaling involving the activation of c-jun N-terminal kinase (JNK) in bovine aortic endothelial cells. A stretch device was designed with the capability of applying cyclic uniaxial and equibiaxial stretches to cultured endothelial cells, as well as changing the direction of cyclic uniaxial stretch. In response to 10% cyclic equibiaxial stretch, which did not result in stress fiber orientation, JNK activation was elevated for up to 6 h. In response to 10% cyclic uniaxial stretch, JNK activity was only transiently elevated, followed by a return to basal level as the actin stress fibers became oriented perpendicular to the direction of stretch. After the stress fibers had aligned perpendicularly and the JNK activity had subsided, a 90-degree change in the direction of cyclic uniaxial stretch reactivated JNK, and this activation again subsided as stress fibers became re-oriented perpendicular to the new direction of stretch. Disrupting actin filaments with cytochalasin D blocked the stress fiber orientation in response to cyclic uniaxial stretch and it also caused the uniaxial stretch-induced JNK activation to become sustained. These results suggest that stress fiber orientation perpendicular to the direction of stretch provides a mechanism for both structural and biochemical adaptation to cyclic mechanical stretch.     

Fiber kinematics of small intestinal submucosa under biaxial and uniaxial stretch

Improving our understanding of the design requirements of biologically derived collagenous scaffolds is necessary for their effective use in tissue reconstruction. In the present study, the collagen fiber kinematics of small intestinal submucosa (SIS) was quantified using small angle light scattering (SALS) while the specimen was subjected to prescribed uniaxial or biaxial strain paths. A modified biaxial stretching device based on Billiar and Sacks (J. Biomech., 30, pp. 753-7, 1997) was used, with a real-time analysis of the fiber kinematics made possible due to the natural translucency of SIS. Results indicated that the angular distribution of collagen fibers in specimens subjected to 10% equibiaxial strain was not significantly different from the initial unloaded condition, regardless of the loading path (p=0.31). Both 10% strip biaxial stretch and uniaxial stretches of greater than 5% in the preferred fiber direction led to an increase in the collagen fiber alignment along the same direction, while 10% strip biaxial stretch in the cross preferred fiber direction led to a broadening of the distribution. While an affine deformation model accurately predicted the experimental findings for a biaxial strain state, uniaxial stretch paths were not accurately predicted. Nonaffine structural models will be necessary to fully predict the fiber kinematics under large uniaxial strains in SIS.     

Computational model predicts cell orientation in response to a range of mechanical stimuli

To build anisotropic, mechanically functioning tissue, it is essential to understand how cells orient in response to mechanical stimuli. Therefore, a computational model was developed which predicts cell orientation, based on the actin stress fiber distribution inside the cell. In the model, the stress fiber distribution evolves dynamically according to the following: (1) Stress fibers contain polymerized actin. The total amount of depolymerized plus polymerized actin is constant. (2) Stress fibers apply tension to their environment. This active tension is maximal when strain rate and absolute strain are zero and reduces with increasing shortening rate and absolute strain. (3) A high active fiber stress in a direction leads to a large amount of fibers in this direction. (4) The cell is attached to a substrate; all fiber stresses are homogenized into a total cell stress, which is in equilibrium with substrate stress. This model predicts that on a substrate of anisotropic stiffness, fibers align in the stiffest direction. Under cyclic strain when the cellular environment is so stiff that no compaction occurs (1 MPa), the model predicts strain avoidance, which is more pronounced with increasing strain frequency or amplitude. Under cyclic strain when the cellular environment is so soft that cells can compact it (10 kPa), the model predicts a preference for the cyclically strained compared to the compacting direction. These model predictions all agree with experimental evidence. For the first time, a computational model predicts cell orientation in response to this range of mechanical stimuli using a single set of parameters.     

Cyclic stretch-induced stress fiber dynamics - Dependence on strain rate, Rho-kinase and MLCK

Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor Y27632 reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.     

Improved prediction of the collagen fiber architecture in the aortic heart valve

Living tissues show an adaptive response to mechanical loading by changing their internal structure and morphology. Understanding this response is essential for successful tissue engineering of load-bearing structures, such as the aortic valve. In this study, mechanically induced remodeling of the collagen architecture in the aortic valve was investigated. It was hypothesized that, in uniaxially loaded regions, the fibers aligned with the tensile principal stretch direction. For biaxial loading conditions, on the other hand, it was assumed that the collagen fibers aligned with directions situated between the principal stretch directions. This hypothesis has already been applied successfully to study collagen remodeling in arteries. The predicted fiber architecture represented a branching network and resembled the macroscopically visible collagen bundles in the native leaflet. In addition, the complex biaxial mechanical behavior of the native valve could be simulated qualitatively with the predicted fiber directions. The results of the present model might be used to gain further insight into the response of tissue engineered constructs during mechanical conditioning.     

周期性单轴拉伸力学刺激对哺乳动物细胞有丝分裂方向影响的研究

哺乳动物细胞的有丝分裂过程与细胞的增殖、分化以及生物体发育、组织器官形成、损伤组织的修复和疾病的发生有关．广泛存在的力学刺激能否对细胞有丝分裂方向产生影响,以及其影响有丝分裂定向的途径尚未完全阐明．采用小鼠成纤维细胞作为模型,研究周期性单轴拉伸力学刺激对细胞应力纤维排布和有丝分裂方向的影响．结果表明,周期性单轴拉伸诱导细胞有丝分裂与应力纤维垂直于拉伸方向排布．而阻断应力纤维的两种基本组成成分(微丝和肌球蛋白Ⅱ),会造成在周期性单轴拉伸条件下的应力纤维和有丝分裂方向重排．特别是,Y27632 (10 μmol/L) 和低浓度的ML7 (50 μmol/L)、Blebbistatin (50 μmol/L)可以诱导细胞有丝分裂与应力纤维平行于拉伸方向排布．统计结果表明,在不同实验条件下,应力纤维排布和有丝分裂方向均具有高度相关性．Western blot实验表明,肌球蛋白轻链磷酸化水平与周期性单轴拉伸刺激下的应力纤维排 布和有丝分裂方向密切相关．上述结果提示：周期性单轴拉伸力学刺激通过诱导应力纤维的排布,决定了细胞的有丝分裂方向．     

A fiber-reinforced composite model of the viscoelastic behavior of the brainstem in shear.

Brainstem trauma occurs frequently in severe head injury, often resulting in fatal lesions due to importance of brainstem in crucial neural functions. Structurally, the brainstem is composed of bundles of axonal fibers distinctly oriented in a longitudinal direction surrounded by an extracellular matrix. We hypothesize that the oriented structure and architecture of the brainstem dictates this mechanical response and results in its selective vulnerability in rotational loading. In order to understand the relationship between the biologic architecture and the mechanical response and provide further insight into the high vulnerability of this region, a structural and mathematical model was created. A fiber-reinforced composite model composed of viscoelastic fibers surrounded by a viscoelastic matrix was used to relate the biological architecture of the brainstem to its anisotropic mechanical response. Relevant model parameters measured include the brainstem's composite complex moduli and relative fraction of matrix and fiber. The model predicted that the fiber component is three times stiffer and more viscous than the matrix. The fiber modulus predictions were compared with experimental tissue measurements. The optic nerve, a bundle of tightly packed longitudinally arranged myelinated fibers with little matrix, served as a surrogate for the brainstem fiber component. Model predictions agreed with experimental measures, offering a validation of the model. This approach provided an understanding of the relationship between the specific biologic architecture of the brainstem and the anisotropic mechanical response and allowed insight into reasons for the selective vulnerability of this region in rotational head injury.     

Experimental Evaluation of Fiber Orientation Based Material Properties of Skeletal Muscle in Tension

Biomechanical researches are essential to develop new techniques to improve the clinical relevance. Skeletal muscle generates the force which results in the motion of human body, so it is essential to study the mechanical and structural properties of skeletal muscle. Many researchers have carried out mechanical study of skeletal muscle with in-vivo testing. This work aims to examine anisotropic mechanical behavior of skeletal muscle with in vitro test (tensile test). It is important to understand the mechanical and structural behavior of skeletal muscle when it is subjected to external loading; the research aims to determine the structural properties of skeletal muscle by tensile testing. Tensile testing is performed on 5 samples of skeletal muscle of a goat at the rate of 1mm/min with fiber orientation along the length and 45° inclined to the length. It is found that muscle is stiffer in the direction parallel to the muscle fiber than at 45° to the muscle fibers. The tensile strength of the skeletal muscle along the fiber direction is 0.44 MPa at maximum load of 110 N and for direction 45° inclined to the muscle fibers, the strength is 0.234 MPa at max load 43 N. The displacement of Muscle sample against the maximum load is small along the length of the muscle fiber i.e. under longitudinal elongation [15.257 mm] as compared to 45° inclined to the length of skeletal muscle [17.775 mm] and under cross fiber elongation [19.7291mm by FEA]. The testing is not performed for 90° fiber orientation due to unavailability of soft tissue in cross fiber direction of the required specification, but finite element analysis is done on the skeletal muscle for the cross fiber orientation. As the fiber orientation within skeletal muscle differs with respect to the length of the muscle, the stiffness of skeletal muscle is also changing effectively. Hence skeletal muscle exhibits the anisotropic mechanical behavior.     

A transversely isotropic viscoelastic constitutive equation for brainstem undergoing finite deformation

The objective of this study was to define the constitutive response of brainstem undergoing finite shear deformation. Brainstem was characterized as a transversely isotropic viscoelastic material and the material model was formulated for numerical implementation. Model parameters were fit to shear data obtained in porcine brainstem specimens undergoing finite shear deformation in three directions: parallel, perpendicular, and cross sectional to axonal fiber orientation and determined using a combined approach of finite element analysis (FEA) and a genetic algorithm (GA) optimizing method. The average initial shear modulus of brainstem matrix of 4-week old pigs was 12.7 Pa, and therefore the brainstem offers little resistance to large shear deformations in the parallel or perpendicular directions, due to the dominant contribution of the matrix in these directions. The fiber reinforcement stiffness was 121.2 Pa, indicating that brainstem is anisotropic and that axonal fibers have an important role in the cross-sectional direction. The first two leading relative shear relaxation moduli were 0.8973 and 0.0741, respectively, with corresponding characteristic times of 0.0047 and 1.4538 s, respectively, implying rapid relaxation of shear stresses. The developed material model and parameter estimation technique are likely to find broad applications in neural and orthopaedic tissues.     

Modeling the effect of collagen fibril alignment on ligament mechanical behavior

Ligament mechanical behavior is primarily regulated by fibrous networks of type I collagen. Although these fibrous networks are typically highly aligned, healthy and injured ligament can also exhibit disorganized collagen architecture. The objective of this study was to determine whether variations in the collagen fibril network between neighboring ligaments can predict observed differences in mechanical behavior. Ligament specimens from two regions of bovine fetlock joints, which either exhibited highly aligned or disorganized collagen fibril networks, were mechanically tested in uniaxial tension. Confocal microscopy and FiberFit software were used to quantify the collagen fibril dispersion and mean fibril orientation in the mechanically tested specimens. These two structural parameters served as inputs into an established hyperelastic constitutive model that accounts for a continuous distribution of planar fibril orientations. The ability of the model to predict differences in the mechanical behavior between neighboring ligaments was tested by (1) curve fitting the model parameters to the stress response of the ligament with highly aligned fibrils and then (2) using this model to predict the stress response of the ligament with disorganized fibrils by only changing the parameter values for fibril dispersion and mean fibril orientation. This study found that when using parameter values for fibril dispersion and mean fibril orientation based on confocal imaging data, the model strongly predicted the average stress response of ligaments with disorganized fibrils (\(R^{2}=0.97\)); however, the model only successfully predicted the individual stress response of ligaments with disorganized fibrils in half the specimens tested. Model predictions became worse when parameters for fibril dispersion and mean fibril orientation were not based on confocal imaging data. These findings emphasize the importance of collagen fibril alignment in ligament mechanics and help advance a mechanistic understanding of fibrillar networks in healthy and injured ligament.     

Hyperelastic properties of human meniscal attachments

Meniscal attachments are ligamentous tissues anchoring the menisci to the underlying subchondral bone. Currently little is known about the behavior of meniscal attachments, with only a few studies quantitatively documenting their properties. The objective of this study was to quantify and compare the tensile mechanical properties of human meniscal attachments in the transverse direction, curve fit experimental Cauchy stress-stretch data to evaluate the hyperelastic behavior, and couple these results with previously obtained longitudinal data to generate a more complete constitutive model. Meniscal attachment specimens were tested using a uniaxial tension test with the collagen fibers oriented perpendicular to the loading axis. Tests were run until failure and load-optical displacement data was recorded for each test. The medial posterior attachment was shown to have a significantly greater elastic modulus (6.42±0.78 MPa) and ultimate stress (1.73±0.32 MPa) when compared to the other three attachments. The Mooney-Rivlin material model was selected as the best fit for the transverse data and used in conjunction with the longitudinal data. A novel computational approach to determining the transition point between the toe and linear regions is presented for the hyperelastic stress-stretch curves. Results from piece-wise non-linear longitudinal curve fitting correlate well with previous linear elastic and SEM findings. These data can be used to advance the design of meniscal replacements and improve knee joint finite element models.     

Evolution of the elastic fiber network of the human uterine cervix before, during and after pregnancy. A quantitative evaluation by automated image analysis

To assess the evolution of the elastic fiber network of the human uterine cervix before, during and after pregnancy, biopsy samples were obtained from 49 women. The high affinity of the polyphenolic compound (+)-catechin for elastin was used to stain the elastic fibers selectively, and enabled automated image analysis. In the human uterine cervix, the elastic fiber network is made up of: (1) fibers running parallel to the basement lamina of the epithelium, and (2) thinner, perpendicular fibers. Quantification using automated image analysis shows a decline in the cervical elastin content from a prepregnancy level of 1.33 +/- 0.08 (SEM) to 0.73 +/- 0.09% (Vv) at the end of pregnancy. In parallel with a constant decline, dissociation and disorganization of the fibers become more clearly evident as pregnancy progresses. However, by 5-7 weeks postpartum the elastic fiber network appears almost completely restructured. These changes support a role of elastin in the processes of cervical maturation and reconstruction during pregnancy and after delivery.     

A kinematic model of stretch-induced stress fiber turnover and reorientation

A kinetic model based on constrained mixture theory was developed to describe the reorganization of actin stress fibers in adherent cells in response to diverse patterns of mechanical stretch. The model was based on reports that stress fibers are pre-extended at a “homeostatic” level under normal, non-perturbed conditions, and that perturbations in stress fiber length destabilize stress fibers. In response to a step change in matrix stretch, the model predicts that stress fibers are initially stretched in registry with the matrix, but that these overly stretched fibers are gradually replaced by new fibers assembled with the homeostatic level of stretch in the new configuration of the matrix. In contrast, average fiber stretch is chronically perturbed from the homeostatic level when the cells are subjected to cyclic equibiaxial stretch. The model was able to describe experimentally measured time courses of stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretch, as well as the lack of alignment in response to equibiaxial stretch. The model also accurately described the relationship between stretch magnitude and the extent of stress fiber alignment in endothelial cells subjected to cyclic uniaxial stretch. Further, in the case of cyclic simple elongation with transverse matrix contraction, stress fibers orient in the direction of least perturbation in stretch. In summary, the model predicts that the rate of stretch-induced stress fiber disassembly determines the rate of alignment, and that stress fibers tend to orient toward the direction of minimum matrix stretch where the rate of stress fiber turnover is a minimum.     

Native extracellular matrix orientation determines multipotent vascular stem cell proliferation in response to cyclic uniaxial tensile strain and simulated stent indentation

Cardiovascular disease is the leading cause of death worldwide, with multipotent vascular stem cells (MVSC) implicated in contributing to diseased vessels. MVSC are mechanosensitive cells which align perpendicular to cyclic uniaxial tensile strain. Within the blood vessel wall, collagen fibers constrain cells so that they are forced to align circumferentially, in the primary direction of tensile strain. In these experiments, MVSC were seeded onto the medial layer of decellularized porcine carotid arteries, then exposed to 10%, 1 Hz cyclic tensile strain for 10 days with the collagen fiber direction either parallel or perpendicular to the direction of strain. Cells aligned with the direction of the collagen fibers regardless of the orientation to strain. Cells aligned with the direction of strain showed an increased number of proliferative Ki67 positive cells, while those strained perpendicular to the direction of cell alignment showed no change in cell proliferation. A bioreactor system was designed to simulate the indentation of a single, wire stent strut. After 10 days of cyclic loading to 10% strain, MVSC showed regions of densely packed, highly proliferative cells. Therefore, MVSC may play a significant role in in-stent restenosis, and this proliferative response could potentially be controlled by controlling MVSC orientation relative to applied strain.     

Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy

Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the beta-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters P2 and P4 of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of P2 and P4 using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the C=O stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the beta-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of P2 and P4 obtained are equal to -0.36 +/- 0.03 and 0.19 +/- 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of P2 and P4 of -0.32 +/- 0.02 and 0.13 +/- 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the beta-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of P2 and P4 using the information entropy theory. For the three types of silk, the beta-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the beta-sheets are nearly Gaussian functions with a width of 32 degrees and 40 degrees for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments.     

Effect of length of the engineered tendon construct on its structure-function relationships in culture

Constructs containing autogenous mesenchymal stem cells (MSCs) seeded in collagen gels have been used by our group to repair rabbit central patellar tendon defect injuries. Although these cell-gel composites exhibit improved repair biomechanics compared to natural healing, they can be difficult to handle at surgery and lack the necessary stiffness to resist peak in vivo forces early thereafter. MSCs are typically suspended in collagen gels around two posts in the base of a well in a specially designed silicone dish. The distance between posts is approximately the length of the tendon wound site. MSCs contract the gel around the posts prior to removal of the construct for implantation at surgery. We hypothesized that in vitro construct alignment and stiffness might be enhanced in the midregion of the longer construct where the end effects of the posts on the bulk material (St. Venant effects) could be minimized. Rabbit MSCs were seeded in purified bovine collagen gel at 0.04 M cells/mg collagen. The cell-gel mixture was pipetted into silicone dishes having two post-to-post lengths (short: 11 mm and long: 51 mm) but equivalent well widths and depths and post diameters. After 14 days of incubation, tensile stiffness and modulus of the constructs were measured using equivalent grip-to-grip lengths. Collagen fiber orientation index or OI (which measures angular dispersion of fibers) was quantified using small angle light scattering (SALS). Long constructs showed significantly lower angular dispersion vs. short constructs (OI of 41.24 degrees +/-1.57 degrees vs. 48.43 degrees +/-1.27 degrees , mean+/-SEM, p<0.001) with significantly higher linear modulus (0.064+/-0.009 MPa vs. 0.024+/-0.004 MPa, p=0.0022) and linear stiffness (0.031+/-0.005 MPa vs. 0.018+/-0.004 N/mm, mean+/-SEM, respectively, p=0.0404). We now plan to use principles of functional tissue engineering to determine if repairs containing central regions of longer MSC-collagen constructs improve defect repair biomechanics after implantation at surgery.