In vitro axial preload application during spine flexibility testing: towards reduced apparatus-related artefacts

Presently, there is little consensus about how, or even if, axial preload should be incorporated in spine flexibility tests in order to simulate the compressive loads naturally present in vivo. Some preload application methods are suspected of producing unwanted “artefact” forces as the specimen rotates and, in doing so, influencing the resulting kinematics. The objective of this study was to quantitatively compare four distinct types of preload which have roots in contemporary experimental practice. The specific quantities compared were the reaction moments and forces resulting at the intervertebral disc and specimen kinematics. The preload types incorporated increasing amounts of caudal constraint on the preload application vector ranging from an unconstrained dead-load arrangement to an apparatus that allowed the vector to follow rotations of the specimen. Six human cadaveric spine segments were tested (1-L1/L2, 3-L2/L3, 1-L3/L4 and 1-L4/L5). Pure moments were applied to the specimens with each of the four different types of compressive preload. Kinematic response was measured using an opto-electronic motion analysis system. A six-axis load cell was used to measure reaction forces and moments. Artefact reaction moments and shear forces were significantly affected by preload application method and magnitude. Unconstrained preload methods produced high artefact moments and low artefact shear forces while more constrained methods did the opposite. A mechanical trade-off is suggested by our results, whereby unwanted moment can only be prevented at the cost of shear force production. When comparing spine flexibility studies, caution should be exercised to ensure preload was applied in a similar manner for all studies. Unwanted moments or forces induced as a result of preload application method may render the comparison of two seemingly similar studies inappropriate.     

Spinal biomechanical properties are significantly altered with a novel embalming method

In vitro tests on the biomechanical properties of human spines are often performed using fresh frozen specimens. However, this carries the risk of pathogen transfer from specimen to the worker and the specimens can only be used for a limited amount of time. Human spinal specimens embalmed with formaldehyde carry an almost absent risk of transfer of pathogens and can be stored and used for a long time, but the tissue properties are strongly affected making this method inapplicable for biomechanical testing. In this study, a new embalming technique called Fix for Life (F4L), which claims to preserve the tissue properties, was tested. The range of motion (ROM) and stiffness of six fresh human spinal specimens was measured using a spinal motion simulator before and after F4L embalming. After F4L embalming, spinal stiffness increased in flexion-extension by 230%, in lateral bending by 284% and in axial rotation by 271%. ROM decreased by 46% in flexion-extension, 56% in lateral bending and 54% in axial rotation. In conclusion, based on this study, F4L does not maintain physiological spinal biomechanical properties, and we propose that this method should not be used for biomechanical studies. Nevertheless, the method may be an alternative to formaldehyde fixation in situations such as training and education because the effect on spinal biomechanics is less detrimental than formaldehyde and tissue color is maintained.     

Leaching of Trace Elements from Biological Tissue by Formalin Fixation

In studies of trace elements in biological tissue, it is imperative that sample handling does not substantially change element concentrations. In many cases, fresh tissue is not available for study, but formalin-fixed tissue is. Formalin fixation has the potential to leach elements from the tissue, but few studies have been published in this area. The concentrations of 19 elements were determined by high-resolution inductively coupled plasma mass spectrometry in formalin in which human and rat brain samples had been stored for different time durations ranging from weeks up to several years. Additional analysis was carried out in fixed brain samples. There was substantial leaching of elements from the tissue into the formalin, and the leaching varied considerably between different elements. For example, formalin concentrations of As, Cd, Mg, Rb, and Sb increased more than 100-fold upon long-term (years) storage, while for Ni and Cr, the leaching was negligible. The degree of leaching was strongly time-dependent. In conclusion, formalin fixation and storage of biological tissue has the potential to leach substantial fractions of several trace elements from the tissue. The potential of leaching must be critically considered when using formalin-fixed biological tissue in trace metal analysis.     

Biomechanical effects of posterior pedicle fixation techniques on the adjacent segment for the treatment of thoracolumbar burst fractures: a biomechanical analysis

Abstract

Posterior pedicle fixation technique is a common method for treating thoracolumbar burst fractures, but the effect of different fixation techniques on the postoperative spinal mechanical properties has not been clearly defined, especially on adjacent segments. A finite element model of T10-L2 with moderate T12 vertebra burst fracture was constructed to investigate biomechanical behavior of three posterior pedicle screw fixation techniques. Compared with traditional short-segment 4 pedicle screw fixation (TS-4) and intermediate long-segment 6 pedicle screw fixation (IL-6), mono-segment 4 pedicle screw fixation (MS-4) provides a safer surgical selection to prevent the secondary degeneration of adjacent segments in the long-term.     

Biaxial biomechanical properties of the nonpregnant murine cervix and uterus

From a biomechanical perspective, female reproductive health is an understudied area of research. There is an incomplete understanding of the complex function and interaction between the cervix and uterus. This, in part, is due to the limited research into multiaxial biomechanical functions and geometry of these organs. Knowledge of the biomechanical function and interaction between these organs may elucidate etiologies of conditions such as preterm birth. Therefore, the objective of this study was to quantify the multiaxial biomechanical properties of the murine cervix and uterus using a biaxial testing set-up. To accomplish this, an inflation-extension testing protocol (n = 15) was leveraged to quantify biaxial biomechanical properties while preserving native matrix interactions and geometry. Ultrasound imaging and histology (n = 10) were performed to evaluate regional geometry and microstructure, respectively. Histological analysis identified a statistically significant greater collagen content and significantly smaller smooth muscle content in the cervix as compared to the uterus. No statistically significant differences in elastic fibers were identified. Analysis of bilinear fits revealed a significantly stiffer response from the circumferentially orientated ECM fibers compared to axially orientated fibers in both organs. Bilinear fits and a two-fiber family constitutive model showed that the cervix was significantly less distensible than the uterus. We submit that the regional biaxial information reported in this study aids in establishing an appropriate reference configuration for mathematical models of the uterine-cervical complex. Thus, may aid future work to elucidate the biomechanical mechanisms leading to cervical or uterine conditions.     

Validation of lumbar spine loading from a musculoskeletal model including the lower limbs and lumbar spine

Low back mechanics are important to quantify to study injury, pain and disability. As in vivo forces are difficult to measure directly, modeling approaches are commonly used to estimate these forces. Validation of model estimates is critical to gain confidence in modeling results across populations of interest, such as people with lower-limb amputation. Motion capture, ground reaction force and electromyographic data were collected from ten participants without an amputation (five male/five female) and five participants with a unilateral transtibial amputation (four male/one female) during trunk-pelvis range of motion trials in flexion/extension, lateral bending and axial rotation. A musculoskeletal model with a detailed lumbar spine and the legs including 294 muscles was used to predict L4-L5 loading and muscle activations using static optimization. Model estimates of L4-L5 intervertebral joint loading were compared to measured intradiscal pressures from the literature and muscle activations were compared to electromyographic signals. Model loading estimates were only significantly different from experimental measurements during trunk extension for males without an amputation and for people with an amputation, which may suggest a greater portion of L4-L5 axial load transfer through the facet joints, as facet loads are not captured by intradiscal pressure transducers. Pressure estimates between the model and previous work were not significantly different for flexion, lateral bending or axial rotation. Timing of model-estimated muscle activations compared well with electromyographic activity of the lumbar paraspinals and upper erector spinae. Validated estimates of low back loading can increase the applicability of musculoskeletal models to clinical diagnosis and treatment.     

Adaptive velocity-based six degree of freedom load control for real-time unconstrained biomechanical testing

Robotic biomechanics is a powerful tool for further developing our understanding of biological joints, tissues and their repair. Both velocity-based and hybrid force control methods have been applied to biomechanics but the complex and non-linear properties of joints have limited these to slow or stepwise loading, which may not capture the real-time behaviour of joints. This paper presents a novel force control scheme combining stiffness and velocity based methods aimed at achieving six degree of freedom unconstrained force control at physiological loading rates.     

Adaptation of a clinical fixation device for biomechanical testing of the lumbar spine

In-vitro biomechanical testing is widely performed for characterizing the load-displacement characteristics of intact, injured, degenerated, and surgically repaired osteoligamentous spine specimens. Traditional specimen fixture devices offer an unspecified rigidity of fixation, while varying in the associated amounts and reversibility of damage to and “coverage” of a specimen – factors that can limit surgical access to structures of interest during testing as well as preclude the possibility of testing certain segments of a specimen. Therefore, the objective of this study was to develop a specimen fixture system for spine biomechanical testing that uses components of clinically available spinal fixation hardware and determine whether the new system provides sufficient rigidity for spine biomechanical testing. Custom testing blocks were mounted into a robotic testing system and the angular deflection of the upper fixture was measured indirectly using linear variable differential transformers. The fixture system had an overall stiffness 37.0, 16.7 and 13.3 times greater than a typical human functional spine unit for the flexion/extension, axial rotation and lateral bending directions respectively – sufficient rigidity for biomechanical testing. Fixture motion when mounted to a lumbar spine specimen revealed average motion of 0.6, 0.6, and 1.5° in each direction. This specimen fixture method causes only minimal damage to a specimen, permits testing of all levels of a specimen, and provides for surgical access during testing.     

福尔马林致痛对大鼠降钙素基因相关肽的影响

目的:研究福尔马林致痛后大鼠脊髓和背根神经节(dorsal root ganglion,DRG)内降钙素基因相关肽(calcitonin gene-related peptide,CGRP)的时空变化规律,为探讨CGRP在伤害性信息传递中的机制和作用提供实验依据。方法:选取健康成年正常SD大鼠54只随机分为生理盐水对照组和福尔马林实验组;实验组为右侧足底皮下给予0.1 mL 5%福尔马林后,分别存活15 min、30min、1 h、3 h、6 h、12h、24h和72h(n=6),免疫组化结合图像分析技术观测CGRP在脊髓腰段及L4~6DRG的表达变化。结果:正常DRG、脊髓前角和后角内CGRP有一定的基础表达。福尔马林致痛后3 h DRG的CGRP表达上升,12 h~24 h达峰值,72 h基本降低到正常;福尔马林致痛后6 h脊髓后角CGRP的表达上调,24 h达到高峰,72 h降低至正常;脊髓前角CGRP未见明显变化。结论:福尔马林致痛引起DRG和脊髓的CGRP的表达呈现一定的时空模式,可能是其参与伤害性信息传递的机制之一。     

Effect of sample treatment on biomechanical properties of insect cuticle

Experimental limitations often prevent to perform biomechanical measurements on fresh arthropod cuticle samples. Hence, in many cases short- or long-term storage of samples is required. So far, it is not known whether any of the standard lab-techniques commonly used to fix or store insect cuticle samples in any way affects the biomechanical properties of the respective samples.In this paper we systematically address this question for the first time, with a focus on practical, easily accessible and common lab-methods including storage in water, ethanol, glutaraldehyde, freezing and desiccation. We performed a comprehensive and sensitive non-destructive Dynamic Mechanical Analysis (DMA) on locust hind leg tibiae using a three-point-bending setup. Our results show that from all tested treatments, freezing samples at −20 °C was the best option to maintain the original values for Young's modulus and damping properties of insect cuticle. In addition, our results indicate that the damping properties of locust hind legs might be mechanically optimized in respect to the jumping and kicking direction.     

Effect of bone fragment impact velocity on biomechanical parameters related to spinal cord injury: A finite element study

Several experimental and computational studies have investigated the effect of bone fragment impact on the spinal cord during trauma. However, the effect of the impact velocity of a fragment generated by a burst fracture on the stress and strain inside the spinal cord has not been computationally investigated, even though spinal canal occlusion and peak pressure at various impact velocities were provided in experimental studies. These stresses and strains are known factors related to clinical symptoms or injuries. In this study, a fluid-structure interaction model of the spinal cord, dura mater, and cerebrospinal fluid was developed and validated. The von-Mises stress distribution in the cord, the longitudinal strain, the cord compression and cross-sectional area at the impact center, and the obliteration of the cerebrospinal fluid layer were analyzed for three pellet sizes at impact velocities ranging from 1.5 m/s to 7.5 m/s. The results indicate that stress in the cord was substantially elevated when the initial impact velocity of the pellet exceeded a threshold of 4.5 m/s. Cord compression, reduction in cross-sectional area, and obliteration of the cerebrospinal fluid increased gradually as the velocity of the pellet increased, regardless of the size of the pellet. The present study provides insight into the mechanisms underlying spinal cord injury.     

Influence of TRAIL gene on biomechanical properties of the human leukemic cell line Jurkat

We cloned the cDNA fragment of human TNF-related apoptosis inducing ligand (TRAIL) into RevTet-On, a Tet-regulated and high-level gene expression system. Making use of the TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the biomechanics properties of Jurkat through measuring changes of cellular biomechanics properties before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the TRAIL gene expression led to significant changes in cellular biomechanics properties. The osmotic fragility increased and the cell stiffness increased after the expression of TRAIL gene. Thus, the apoptosis-inducing TRAIL gene caused significant changes in the biomechanics properties of Jurkat cells.     

体外苏氨酸循环固碳途径的构建

乙酰CoA是生物体代谢过程中重要的代谢物,也是许多有价值产品合成的前体物质。然而传统途径中通过丙酮酸脱羧生成乙酰CoA碳得率较低,因此构建一条高效的乙酰CoA合成途径具有重要的意义。由于在体外验证文献报道的高碳摩尔得率合成乙酰CoA的苏氨酸循环固碳途径,有较重要的理论意义和应用价值。因此在体外构建了苏氨酸循环固碳途径合成乙酰CoA,通过分段加酶的方式将其在体外进行了验证。在体外验证时,以丙酮酸为底物,则丝氨酸脱氨酶(Tdc B)为循环途径的最后一步反应。结果表明,当加入途径中除丝氨酸脱氨酶之外的酶时,测得的乙酰CoA浓度约1.5 mmol/L,待反应达到平衡时,加入丝氨酸脱氨酶,丝氨酸转化为丙酮酸,丙酮酸再次进入循环,乙酰CoA的量增加了约0.2 mmol/L,由此得出结论在体外苏氨酸循环实现了固碳。     

A biomechanical comparison of composite femurs and cadaver femurs used in experiments on operated hip fractures

Fourth generation composite femurs (4GCFs, models #3406 and #3403) simulate femurs of males <80 years with good bone quality. Since most hip fractures occur in old women with fragile bones, concern is raised regarding the use of standard 4GCFs in biomechanical experiments. In this study the stability of hip fracture fixations in 4GCFs was compared to human cadaver femurs (HCFs) selected to represent patients with hip fractures.Ten 4GCFs (Sawbones, Pacific Research Laboratories, Inc., Vashon, WA, USA) were compared to 24 HCFs from seven females and five males >60 years. Proximal femur anthropometric measurements were noted. Strain gauge rosettes were attached and femurs were mounted in a hip simulator applying a combined subject-specific axial load and torque. Baseline measurements of resistance to deformation were recorded. Standardized femoral neck fractures were surgically stabilized before the constructs were subjected to 20,000 load-cycles. An optical motion tracking system measured relative movements.Median (95% CI) head fragment migration was 0.8 mm (0.4 to 1.1) in the 4GCF group versus 2.2 mm (1.5 to 4.6) in the cadaver group (p=0.001). This difference in fracture stability could not be explained by observed differences in femoral anthropometry or potential overloading of 4GCFs. 4GCFs failed with fracture-patterns different from those observed in cadavers.To conclude, standard 4GCFs provide unrealistically stable bone-implant constructs and fail with fractures not observed in cadavers. Until a validated osteopenic or osteoporotic composite femur model is provided, standard 4GCFs should only be used when representing the biomechanical properties of young healthy femurs.     

Freezing for postmortal storage influences the biomechanical properties of linear skin wounds.

Specimens for biomechanical investigations are often stored frozen between sampling and testing. Several authors have analysed the effects of freezing on a variety of intact tissues; while some have found mostly minor changes, others have reported no adverse effects. Healing wounds contain more fragile tissue components than other tissues and are therefore more sensitive to possible adverse effects. This study on rat skin wounds (healed for 10 and 20 days) demonstrates that freezing has a significant adverse influence on the mechanical properties. It is concluded that fresh tissue should be used whenever possible. In case storage in a freezer is necessary great care should be taken when designing the experimental protocol.     

Multiaxial mechanical behavior of the porcine anterior lens capsule

The biomechanics of the lens capsule of the eye is important both in physiologic processes such as accommodation and clinical treatments such as cataract surgery. Although the lens capsule experiences multiaxial stresses in vivo, there have been no measurements of its multiaxial properties or possible regional heterogeneities. Rather all prior mechanical data have come from 1-D pressure–volume or uniaxial force-length tests. Here, we report a new experimental approach to study in situ the regional, multiaxial mechanical behavior of the lens capsule. Moreover, we report multiaxial data suggesting that the porcine anterior lens capsule exhibits a typical nonlinear pseudoelastic behavior over finite strains, that the in situ state is pre-stressed multiaxially, and that the meridional and circumferential directions are principal directions of strain, which is nearly equibiaxial at the pole but less so towards the equator. Such data are fundamental to much needed constitutive formulations.     

Increase in facet joint loading after nucleotomy in the human lumbar spine

Low-back pain has been related to degenerative changes after nucleotomy. Although several etiologies for pain after nucleotomy have been proposed, there is evidence of pain arising in the facet joints in general, which may be related to changes in load transfer. This study addresses the effect of nucleotomy on facet joint loading.     

Effects of freeze-thaw on the biomechanical and structural properties of the rat Achilles tendon

Rodent models are commonly used to investigate tendon healing, with the biomechanical and structural properties of the healed tendons being important outcome measures. Tendon storage for later testing becomes necessary when performing large experiments with multiple time-points. However, it is unclear whether freezing rodent tendons affects their material properties. Thus the aim of this study was to determine whether freezing rat Achilles tendons affects their biomechanical or structural properties. Tendons were frozen at either −20 °C or −80 °C directly after harvesting, or tested when freshly harvested. Groups of tendons were subjected to several freeze-thaw cycles (1, 2, and 5) within 3 months, or frozen for 9 months, after which the tendons were subjected to biomechanical testing. Additionally, fresh and thawed tendons were compared morphologically, histologically and by transmission electron microscopy. No major differences in biomechanical properties were found between fresh tendons and those frozen once or twice at −20 °C or −80 °C. However, deterioration of tendon properties was found for 5-cycle groups and both long-term freezing groups; after 9 months of freezing at −80 °C the tear resistance of the tendon was reduced from 125.4 ± 16.4N to 74.3 ± 18.4N (p = 0.0132). Moreover, tendons stored under these conditions showed major disruption of collagen fibrils when examined by transmission electron microscopy. When examined histologically, fresh samples exhibited the best cellularity and proteoglycan content of the enthesis. These properties were preserved better after freezing at −80 °C than after freezing at −20 °C, which resulted in markedly smaller chondrocytes and less proteoglycan content. Overall, the best preservation of histological integrity was seen with tendons frozen once at −80 °C. In conclusion, rat Achilles tendons can be frozen once or twice for short periods of time (up to 3 months) at −20 °C or −80 °C for later testing. However, freezing for 9 months at either −20 °C or −80 °C leads to deterioration of certain parameters.     

Cancer cells regulate biomechanical properties of human microvascular endothelial cells

Metastasis is a key event of malignant tumor progression. The capability to metastasize depends on the ability of the cancer cell to migrate into connective tissue, adhere, and possibly transmigrate through the endothelium. Previously we reported that the endothelium does not generally act as barrier for cancer cells to migrate in three-dimensional extracellular matrices (3D-ECMs). Instead, the endothelium acts as an enhancer or a promoter for the invasiveness of certain cancer cells. How invasive cancer cells diminish the endothelial barrier function still remains elusive. Therefore, this study investigates whether invasive cancer cells can decrease the endothelial barrier function through alterations of endothelial biomechanical properties. To address this, MDA-MB-231 breast cancer cells were used that invade deeper and more numerous into 3D-ECMs when co-cultured with microvascular endothelial cells. Using magnetic tweezer measurements, MDA-MB-231 cells were found to alter the mechanical properties of endothelial cells by reducing endothelial cell stiffness. Using spontaneous bead diffusion, actin cytoskeletal remodeling dynamics were shown to be increased in endothelial cells co-cultured with MDA-MB-231 cells compared with mono-cultured endothelial cells. In addition, knockdown of the α5 integrin subunit in highly transmigrating α5β1(high) cells derived from breast, bladder, and kidney cancer cells abolished the endothelial invasion-enhancing effect comparable with the inhibition of myosin light chain kinase. These results indicate that the endothelial invasion-enhancing effect is α5β1 integrin-dependent. Moreover, inhibition of Rac-1, Rho kinase, MEK kinase, and PI3K reduced the endothelial invasion-enhancing effect, indicating that signaling via small GTPases may play a role in the endothelial facilitated increased invasiveness of cancer cells. In conclusion, decreased stiffness and increased cytoskeletal remodeling dynamics of endothelial cells may account for the breakdown of endothelial barrier function, suggesting that biomechanical alterations are sufficient to facilitate the transmigration and invasion of invasive cancer cells into 3D-ECMs.