Review of state-of-the-art micro and macro-bioreactors for the intervertebral disc.

Lower back pain continues to be a global epidemic, limiting quality of life and ability to work, due in large part to symptomatic disc degeneration. Development of more effective and less invasive biological strategies are needed to treat disc degeneration. In vitro models such as macro- or micro-bioreactors or mechanically active organ-chips hold great promise in reducing the need for animal studies that may have limited clinical translatability, due to harsher and more complex mechanical loading environments in human discs than in most animal models. This review highlights the complex loading conditions of the disc in situ, evaluates state-of-the-art designs for applying such complex loads across multiple length scales, from macro-bioreactors that load whole discs to organ-chips that aim to replicate cellular or engineered tissue loading. Emphasis was placed on the rapidly evolving more customizable organ-chips, given their greater potential for studying the progression and treatment of symptomatic disc degeneration. Lastly, this review identifies new trends and challenges for using organ-chips to assess therapeutic strategies.

Effect of Passaging on Bovine Chondrocyte Gene Expression and Engineered Cartilage Production.

Tissue engineering strategies show great potential for repairing osteochondral defects in osteoarthritic joints; however, these approaches often rely on passaging cells multiple times to obtain enough cells to produce functional tissue. Unfortunately, monolayer expansion culture causes chondrocyte dedifferentiation, which is accompanied by a phenotypical and morphological shift in chondrocyte properties that leads to a reduction in the quality of de novo cartilage produced. Thus, the objective of this study was to evaluate transcriptional variations during in vitro expansion culture and determine how differences in cell phenotype from monolayer expansion alter development of functional engineered cartilage. We used an unbiased approach to explore genome-wide transcriptional differences in chondrocyte phenotype at passage 1 (P1), P3, and P5, and then seeded cells into hydrogel scaffolds at P3 and P5 to assess cells' abilities to produce cartilaginous extracellular matrix in three dimensional (3D). We identified distinct phenotypic differences, specifically for genes related to extracellular organization and cartilage development. Both P3 and P5 chondrocytes were able to produce chondrogenic tissue in 3D, with P3 cells producing matrix with greater compressive properties and P5 cells secreting matrix with higher glycosaminoglycan/DNA and collagen/DNA ratios. Furthermore, we identified 24 genes that were differentially expressed with passaging and enriched in human osteoarthritis (OA) genome-wide association studies, thereby prioritizing them as functionally relevant targets to improve protocols that recapitulate functional healthy cartilage with cells from adult donors. Specifically, we identified novel genes, such as TMEM190 and RAB11FIP4, which were enriched with human hip OA and may play a role in chondrocyte dedifferentiation. This work lays the foundation for several pathways and genes that could be modulated to enhance the efficacy for chondrocyte culture for tissue regeneration, which could have transformative impacts for cell-based cartilage repair strategies.

A Systems Approach to Biomechanics, Mechanobiology, and Biotransport.

The human body represents a collection of interacting systems that range in scale from nanometers to meters. Investigations from a systems perspective focus on how the parts work together to enact changes across spatial scales, and further our understanding of how systems function and fail. Here, we highlight systems approaches presented at the 2022 Summer Biomechanics, Bio-engineering, and Biotransport Conference in the areas of solid mechanics; fluid mechanics; tissue and cellular engineering; biotransport; and design, dynamics, and rehabilitation; and biomechanics education. Systems approaches are yielding new insights into human biology by leveraging state-of-the-art tools, which could ultimately lead to more informed design of therapies and medical devices for preventing and treating disease as well as rehabilitating patients using strategies that are uniquely optimized for each patient. Educational approaches can also be designed to foster a foundation of systems-level thinking.

Understanding the etiopathogenesis of lumbar intervertebral disc herniation: From clinical evidence to basic scientific research.

Lumbar intervertebral disc herniation, as a leading cause of low back pain, productivity loss, and disability, is a common musculoskeletal disorder that results in significant socioeconomic burdens. Despite extensive clinical and basic scientific research efforts, herniation etiopathogenesis, particularly its initiation and progression, is not well understood. Understanding herniation etiopathogenesis is essential for developing effective preventive measures and therapeutic interventions. Thus, this review seeks to provide a thorough overview of the advances in herniation-oriented research, with a discussion on ongoing challenges and potential future directions for clinical, translational, and basic scientific investigations to facilitate innovative interdisciplinary research aimed at understanding herniation etiopathogenesis. Specifically, risk factors for herniation are identified and summarized, including familial predisposition, obesity, diabetes mellitus, smoking tobacco, selected cardiovascular diseases, disc degeneration, and occupational risks. Basic scientific experimental and computational research that aims to understand the link between excessive mechanical load, catabolic tissue remodeling due to inflammation or insufficient nutrient supply, and herniation, are also reviewed. Potential future directions to address the current challenges in herniation-oriented research are explored by combining known progressive development in existing research techniques with ongoing technological advances. More research on the relationship between occupational risk factors and herniation, as well as the relationship between degeneration and herniation, is needed to develop preventive measures for working-age individuals. Notably, researchers should explore using or modifying existing degeneration animal models to study herniation etiopathogenesis, as such models may allow for a better understanding of how to prevent mild-to-moderately degenerated discs from herniating.

Priming chondrocytes during expansion alters cell behavior and improves matrix production in 3D culture.

OBJECTIVE: Cartilage tissue engineering strategies that use autologous chondrocytes require in vitro expansion of cells to obtain enough cells to produce functional engineered tissue. However, chondrocytes dedifferentiate during expansion culture, limiting their ability to produce chondrogenic tissue and their utility for cell-based cartilage repair strategies. The current study identified conditions that favor cartilage production and the mechanobiological mechanisms responsible for these benefits. DESIGN: Chondrocytes were isolated from juvenile bovine knee joints and cultured with (primed) or without (unprimed) a growth factor cocktail. Gene expression, cell morphology, cell adhesion, cytoskeletal protein distribution, and cell mechanics were assessed. Following passage 5, cells were embedded into agarose hydrogels to evaluate functional properties of engineered cartilage. RESULTS: Priming cells during expansion culture altered cell phenotype and chondrogenic tissue production. Unbiased ribonucleic acid-sequencing analysis suggested, and experimental studies confirmed, that growth factor priming delays dedifferentiation associated changes in cell adhesion and cytoskeletal organization. Priming also overrode mechanobiological pathways to prevent chondrocytes from remodeling their cytoskeleton to accommodate the stiff, monolayer microenvironment. Passage 1 primed cells deformed less and had lower yes associated protein 1 activity than unprimed cells. Differences in cell adhesion, morphology, and cell mechanics between primed and unprimed cells were mitigated by passage 5. CONCLUSIONS: Priming suppresses mechanobiologic cytoskeletal remodeling to prevent chondrocyte dedifferentiation, resulting in more cartilage-like tissue-engineered constructs.

Non-enzymatic glycation increases the failure risk of annulus fibrosus by predisposing the extrafibrillar matrix to greater stresses.

Growing clinical evidence suggests a correlation between diabetes and more frequent and severe intervertebral disc failure, partially attributed to accelerated advanced glycation end-products (AGE) accumulation in the annulus fibrosus (AF) through non-enzymatic glycation. However, in vitro glycation (i.e., crosslinking) reportedly improved AF uniaxial tensile mechanical properties, contradicting clinical observations. Thus, this study used a combined experimental-computational approach to evaluate the effect of AGEs on anisotropic AF tensile mechanics, applying finite element models (FEMs) to complement experimental testing and examine difficult-to-measure subtissue-level mechanics. Methylglyoxal-based treatments were applied to induce three physiologically relevant AGE levels in vitro. Models incorporated crosslinks by adapting our previously validated structure-based FEM framework. Experimental results showed that a threefold increase in AGE content resulted in a ∼55% increase in AF circumferential-radial tensile modulus and failure stress and a 40% increase in radial failure stress. Failure strain was unaffected by non-enzymatic glycation. Adapted FEMs accurately predicted experimental AF mechanics with glycation. Model predictions showed that glycation increased stresses in the extrafibrillar matrix under physiologic deformations, which may increase tissue mechanical failure or trigger catabolic remodeling, providing insight into the relationship between AGE accumulation and increased tissue failure. Our findings also added to the existing literature regarding crosslinking structures, indicating that AGEs had a greater effect along the fiber direction, while interlamellar radial crosslinks were improbable in the AF. In summary, the combined approach presented a powerful tool for examining multiscale structure-function relationships with disease progression in fiber-reinforced soft tissues, which is essential for developing effective therapeutic measures. STATEMENT OF SIGNIFICANCE: Increasing clinical evidence correlates diabetes with premature intervertebral disc failure, likely due to advanced glycation end-products (AGE) accumulation in the annulus fibrosus (AF). However, in vitro glycation reportedly increases AF tensile stiffness and toughness, contradicting clinical observations. Using a combined experimental-computational approach, our work shows that increases in AF bulk tensile mechanical properties with glycation are achieved at the risk of exposing the extrafibrillar matrix to increased stresses under physiologic deformations, which may increase tissue mechanical failure or trigger catabolic remodeling. Computational results indicate that crosslinks along the fiber direction account for 90% of the increased tissue stiffness with glycation, adding to the existing literature. These findings provide insight into the multiscale structure-function relationship between AGE accumulation and tissue failure.

Design of a flexing organ-chip to model in situ loading of the intervertebral disc.

The leading cause of disability of all ages worldwide is severe lower back pain. To address this untreated epidemic, further investigation is needed into the leading cause of back pain, intervertebral disc degeneration. In particular, microphysiological systems modeling critical tissues in a degenerative disc, like the annulus fibrosus (AF), are needed to investigate the effects of complex multiaxial strains on AF cells. By replicating these mechanobiological effects unique to the AF that are not yet understood, we can advance therapies for early-stage degeneration at the cellular level. To this end, we designed, fabricated, and collected proof-of-concept data for a novel microphysiological device called the flexing annulus-on-a-chip (AoC). We used computational models and experimental measurements to characterize the device's ability to mimic complex physiologically relevant strains. As a result, these strains proved to be controllable, multi-directional, and uniformly distributed with magnitudes ranging from - 10 % to 12% in the axial, radial, and circumferential directions, which differ greatly from applied strains possible in uniaxial devices. Furthermore, after withstanding accelerated life testing (66 K cycles of 10% strain) and maintaining 2000 bovine AF cells without loading for more than three weeks the AoC proved capable of long-term cell culture. Additionally, after strain (3.5% strain for 75 cycles at 0.5 Hz) was applied to a monolayer of AF cells in the AoC, a population remained adhered to the channel with spread morphology. The AoC can also be tailored for other annular structures in the body such as cardiovascular vessels, lymphatic vessels, and the cervix.

Disc geometry measurement methods affect reported compressive mechanics by up to 65.

Mechanical testing is a valuable tool for assessing intervertebral disc health, but the wide range of testing protocols makes it difficult to compare results from different studies. Normalizing mechanical properties by disc geometry allows for such comparisons, but there is little consistency in the methods by which disc geometry is measured. As such, we hypothesized that methods used to measure disc geometry would impact reported mechanical properties. Disc height and area were measured using computed tomography (CT), digital calipers, and ImageJ to yield three different measurements for disc height and six for disc area. Disc heights measured by digital calipers ex situ were >30% less than disc heights measured in situ by CT, and disc areas measured ex situ using ImageJ were >30% larger than those measured by CT. This significantly affected reported mechanical properties, leading to a 65% reduction in normalized compressive stiffness in the most extreme case. Though we cannot quantitatively correct between methods, results presented in this study suggest that disc geometry measurement methods have a significant impact on normalized mechanical properties and should be accounted for when comparing results.

Addition of collagen type I in agarose created a dose-dependent effect on matrix production in engineered cartilage.

Extracellular-matrix composition impacts mechanical performance in native and engineered tissues. Previous studies showed collagen type I-agarose blends increased cell-matrix interactions and extracellular matrix production. However, long-term impacts on protein production and mechanical properties of engineered cartilage are unknown. Our objective was to characterize the effect of collagen type I on the matrix production of chondrocytes embedded in agarose hydrogels. We hypothesized that the addition of collagen would improve long-term mechanical properties and matrix production (e.g. collagen and glycosaminoglycans) through increased bioactivity. Agarose hydrogels (2% w/v) were mixed with varying concentrations of collagen type I (0, 2 and 5 mg/ml). Juvenile bovine chondrocytes were added to the hydrogels to assess matrix production over 4 weeks through biochemical assays, and mechanical properties were assessed through unconfined compression. We observed a dose-dependent effect on cell bioactivity, where 2 mg/ml of collagen improved bioactivity, but 5 mg/ml had a negative impact on bioactivity. This resulted in a higher modulus for scaffolds supplemented with lower collagen concentration as compared to the higher collagen concentration, but not when compared to the control. In conclusion, the addition of collagen to agarose constructs provided a dose-dependent impact on improving glycosaminoglycan production but did not improve collagen production or compressive mechanics.

Torque- and Muscle-Driven Flexion Induce Disparate Risks of In Vitro Herniation: A Multiscale and Multiphasic Structure-Based Finite Element Study.

The intervertebral disc is a complex structure that experiences multiaxial stresses regularly. Disc failure through herniation is a common cause of lower back pain, which causes reduced mobility and debilitating pain, resulting in heavy socioeconomic burdens. Unfortunately, herniation etiology is not well understood, partially due to challenges in replicating herniation in vitro. Previous studies suggest that flexion elevated risks of herniation. Thus, the objective of this study was to use a multiscale and multiphasic finite element model to evaluate the risk of failure under torque- or muscle-driven flexion. Models were developed to represent torque-driven flexion with the instantaneous center of rotation (ICR) located on the disc, and the more physiologically representative muscle-driven flexion with the ICR located anterior of the disc. Model predictions highlighted disparate disc mechanics regarding bulk deformation, stress-bearing mechanisms, and intradiscal stress-strain distributions. Specifically, failure was predicted to initiate at the bone-disc boundary under torque-driven flexion, which may explain why endplate junction failure, instead of herniation, has been the more common failure mode observed in vitro. By contrast, failure was predicted to initiate in the posterolateral annulus fibrosus under muscle-driven flexion, resulting in consistent herniation. Our findings also suggested that muscle-driven flexion combined with axial compression could be sufficient for provoking herniation in vitro and in silico. In conclusion, this study provided a computational framework for designing in vitro testing protocols that can advance the assessment of disc failure behavior and the performance of engineered disc implants.

Saline-polyethylene glycol blends preserve in vitro annulus fibrosus hydration and mechanics: An experimental and finite-element analysis.

Precise control of tissue water content is essential for ensuring accurate, repeatable, and physiologically relevant measurements of tissue mechanics and biochemical composition. While previous studies have found that saline and polyethylene glycol (PEG) blends were effective at controlling tendon and ligament hydration levels, this work has yet to be extended to the annulus fibrosus (AF). Thus, the first objective of this study was to determine and validate an optimal buffer solution for targeting and maintaining hydration levels of tissue-level AF specimens in vitro. This was accomplished by measuring the transient swelling behavior of bovine AF specimens in phosphate-buffered saline (PBS) and PEG buffers across a wide range of concentrations. Sub-failure, failure, and post-failure mechanics were measured to determine the relationship between changes in tissue hydration and tensile mechanical response. The effect of each buffer solution on tissue composition was also assessed. The second objective of this study was to assess the feasibility and effectiveness of using multi-phasic finite element models to investigate tissue swelling and mechanical responses in different external buffer solutions. A solution containing 6.25%w/v PBS and 6.25%w/v PEG effectively maintained tissue-level AF specimen hydration at fresh-frozen levels after 18 h in solution. Modulus, failure stress, failure strain, and post-failure toughness of specimens soaked in this solution for 18 h closely matched those of fresh-frozen specimens. In contrast, specimens soaked in 0.9%w/v PBS swelled over 100% after 18 h and exhibited significantly diminished sub-failure and failure properties compared to fresh-frozen controls. The increased cross-sectional area with swelling contributed to but was not sufficient to explain the diminished mechanics of PBS-soaked specimens, suggesting additional sub-tissue scale mechanisms. Computational simulations of these specimens generally agreed with experimental results, highlighting the feasibility and importance of including a fluid-phase description when models aim to provide accurate predictions of biological tissue responses. As numerous previous studies suggest that tissue hydration plays a central role in maintaining proper mechanical and biological function, robust methods for controlling hydration levels are essential as the field advances in probing the relationship between tissue hydration, aging, injury, and disease.

Non-enzymatic glycation of annulus fibrosus alters tissue-level failure mechanics in tension.

Advanced-glycation end products (AGEs) are known to accumulate in biological tissues with age and at an accelerated rate in patients with diabetes and chronic kidney disease. Clinically, diabetes has been linked to increased frequency and severity of back pain, accelerated disc degeneration, and an increased risk of disc herniation. Despite significant clinical evidence suggesting that diabetes-induced AGEs may play a role in intervertebral disc failure and substantial previous work investigating the effects of AGEs on bone, cartilage, and tendon mechanics, the effects of AGEs on annulus fibrosus (AF) failure mechanics have not yet been reported. Thus, the aim of this study was to determine the relationship between physiological levels of AGEs and AF tensile mechanics at two distinct loading rates. In vitro glycation treatments with methylglyoxal were applied to minimize changes in tissue hydration and induce two distinct levels of AGEs based on values measured from human AF tissues. In vitro glycation increased modulus by 48-99% and failure stress by 45-104% versus control and decreased post-failure energy absorption capacity by 15-32% versus control (ANOVA p < 0.0001 on means; range given across two loading rates and glycation levels). AGE content correlated strongly with modulus (R = 0.74, p < 0.0001) and failure stress (R = 0.70, p < 0.0001) and moderately with post-failure energy absorption capacity (R = 0.62, p < 0.0001). Failure strain was reduced by 10-17% at the high-glycation level (ANOVA p = 0.01). Tissue water content remained near or just above fresh-tissue levels for all groups. The alterations in mechanics with glycation reported here are consistent with trends from other connective tissues but do not fully explain the clinical predisposition of diabetics to disc herniation. The results from this study may be used in the development of advanced computational models that aim to study disc disease progression and to provide a deeper understanding of altered structure-function relationships that may lead to tissue dysfunction and failure with aging and disease.

Relations Between Bone Quantity, Microarchitecture, and Collagen Cross-links on Mechanics Following In Vivo Irradiation in Mice.

Humans are exposed to ionizing radiation via spaceflight or cancer radiotherapy, and exposure from radiotherapy is known to increase risk of skeletal fractures. Although irradiation can reduce trabecular bone mass, alter trabecular microarchitecture, and increase collagen cross-linking, the relative contributions of these effects to any loss of mechanical integrity remain unclear. To provide insight, while addressing both the monotonic strength and cyclic-loading fatigue life, we conducted total-body, acute, gamma-irradiation experiments on skeletally mature (17-week-old) C57BL/6J male mice (n = 84). Mice were administered doses of either 0 Gy (sham), 1 Gy (motivated by cumulative exposures from a Mars mission), or 5 Gy (motivated by clinical therapy regimens) with retrieval of the lumbar vertebrae at either a short-term (11-day) or long-term (12-week) time point after exposure. Micro-computed tomography was used to assess trabecular and cortical quantity and architecture, biochemical composition assays were used to assess collagen quality, and mechanical testing was performed to evaluate vertebral compressive strength and fatigue life. At 11 days post-exposure, 5 Gy irradiation significantly reduced trabecular mass (p < 0.001), altered microarchitecture (eg, connectivity density p < 0.001), and increased collagen cross-links (p < 0.001). Despite these changes, vertebral strength (p = 0.745) and fatigue life (p = 0.332) remained unaltered. At 12 weeks after 5 Gy exposure, the trends in trabecular bone persisted; in addition, regardless of irradiation, cortical thickness (p < 0.01) and fatigue life (p < 0.01) decreased. These results demonstrate that the highly significant effects of 5 Gy total-body irradiation on the trabecular bone morphology and collagen cross-links did not translate into detectable effects on vertebral mechanics. The only mechanical deficits observed were associated with aging. Together, these vertebral results suggest that for spaceflight, irradiation alone will likely not alter failure properties, and for radiotherapy, more investigations that include post-exposure time as a positive control and testing of both failure modalities are needed to determine the cause of increased fracture risk. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

A Robust Multiscale and Multiphasic Structure-Based Modeling Framework for the Intervertebral Disc.

A comprehensive understanding of multiscale and multiphasic intervertebral disc mechanics is crucial for designing advanced tissue engineered structures aiming to recapitulate native tissue behavior. The bovine caudal disc is a commonly used human disc analog due to its availability, large disc height and area, and similarities in biochemical and mechanical properties to the human disc. Because of challenges in directly measuring subtissue-level mechanics, such as in situ fiber mechanics, finite element models have been widely employed in spinal biomechanics research. However, many previous models use homogenization theory and describe each model element as a homogenized combination of fibers and the extrafibrillar matrix while ignoring the role of water content or osmotic behavior. Thus, these models are limited in their ability in investigating subtissue-level mechanics and stress-bearing mechanisms through fluid pressure. The objective of this study was to develop and validate a structure-based bovine caudal disc model, and to evaluate multiscale and multiphasic intervertebral disc mechanics under different loading conditions and with degeneration. The structure-based model was developed based on native disc structure, where fibers and matrix in the annulus fibrosus were described as distinct materials occupying separate volumes. Model parameters were directly obtained from experimental studies without calibration. Under the multiscale validation framework, the model was validated across the joint-, tissue-, and subtissue-levels. Our model accurately predicted multiscale disc responses for 15 of 16 cases, emphasizing the accuracy of the model, as well as the effectiveness and robustness of the multiscale structure-based modeling-validation framework. The model also demonstrated the rim as a weak link for disc failure, highlighting the importance of keeping the cartilage endplate intact when evaluating disc failure mechanisms in vitro. Importantly, results from this study elucidated important fluid-based load-bearing mechanisms and fiber-matrix interactions that are important for understanding disease progression and regeneration in intervertebral discs. In conclusion, the methods presented in this study can be used in conjunction with experimental work to simultaneously investigate disc joint-, tissue-, and subtissue-level mechanics with degeneration, disease, and injury.

Spine biomechanical testing methodologies: The controversy of consensus vs scientific evidence.

Biomechanical testing methodologies for the spine have developed over the past 50 years. During that time, there have been several paradigm shifts with respect to techniques. These techniques evolved by incorporating state-of-the-art engineering principles, in vivo measurements, anatomical structure-function relationships, and the scientific method. Multiple parametric studies have focused on the effects that the experimental technique has on outcomes. As a result, testing methodologies have evolved, but there are no standard testing protocols, which makes the comparison of findings between experiments difficult and conclusions about in vivo performance challenging. In 2019, the international spine research community was surveyed to determine the consensus on spine biomechanical testing and if the consensus opinion was consistent with the scientific evidence. More than 80 responses to the survey were received. The findings of this survey confirmed that while some methods have been commonly adopted, not all are consistent with the scientific evidence. This review summarizes the scientific literature, the current consensus, and the authors' recommendations on best practices based on the compendium of available evidence.

Intervertebral Disc Mechanics With Nucleotomy: Differences Between Simple and Dual Loading.

Painful herniated discs are treated surgically by removing extruded nucleus pulposus (NP) material (nucleotomy). NP removal through enzymatic digestion is also commonly performed to initiate degenerative changes to study potential biological repair strategies. Experimental and computational studies have shown a decrease in disc stiffness with nucleotomy under single loading modalities, such as compression-only or bending-only loading. However, studies that apply more physiologically relevant loading conditions, such as compression in combination with bending or torsion, have shown contradicting results. We used a previously validated bone-disc-bone finite element model (Control) to create a Nucleotomy model to evaluate the effect of dual loading conditions (compression with torsion or bending) on intradiscal deformations. While disc joint stiffness decreased with nucleotomy under single loading conditions, as commonly reported in the literature, dual loading resulted in an increase in bending stiffness. More specifically, dual loading resulted in a 40% increase in bending stiffness under flexion and extension and a 25% increase in stiffness under lateral bending. The increase in bending stiffness was due to an increase and shift in compressive stress, where peak stresses migrated from the NP-annulus interface to the outer annulus. In contrast, the decrease in torsional stiffness was due to greater fiber reorientation during compression. In general, large radial strains were observed with nucleotomy, suggesting an increased risk for delamination or degenerative remodeling. In conclusion, the effect of nucleotomy on disc mechanics depends on the type and complexity of applied loads.

Fiber engagement accounts for geometry-dependent annulus fibrosus mechanics: A multiscale, Structure-Based Finite Element Study.

A comprehensive understanding of biological tissue mechanics is crucial for designing engineered tissues that aim to recapitulate native tissue behavior. Tensile mechanics of many fiber-reinforced tissues have been shown to depend on specimen geometry, which makes it challenging to compare data between studies. In this study, a validated multiscale, structure-based finite element model was used to evaluate the effect of specimen geometry on multiscale annulus fibrosus tensile mechanics through a fiber engagement analysis. The relationships between specimen geometry and modulus, Poisson's ratio, tissue stress-strain distributions, and fiber reorientation behaviors were investigated at both tissue and sub-tissue levels. It was observed that annulus fibrosus tissue level tensile properties and stress transmission mechanisms were dependent on specimen geometry. The model also demonstrated that the contribution of fiber-matrix interactions to tissue mechanical response was specimen size- and orientation-dependent. The results of this study reinforce the benefits of structure-based finite element modeling in studies investigating multiscale tissue mechanics. This approach also provides guidelines for developing optimal combined computational-experimental study designs for investigating fiber-reinforced biological tissue mechanics. Additionally, findings from this study help explain the geometry dependence of annulus fibrosus tensile mechanics previously reported in the literature, providing a more fundamental and comprehensive understanding of tissue mechanical behavior. In conclusion, the methods presented here can be used in conjunction with experimental tissue level data to simultaneously investigate tissue and sub-tissue scale mechanics, which is important as the field of soft tissue biomechanics advances toward studies that focus on diminishing length scales.

Influence of testing environment and loading rate on intervertebral disc compressive mechanics: An assessment of repeatability at three different laboratories.

In vitro mechanical testing of intervertebral discs is crucial for basic science and pre-clinical testing. Generally, these tests aim to replicate in vivo conditions, but simplifications are necessary in specimen preparation and mechanical testing due to complexities in both structure and the loading conditions required to replicate in vivo conditions. There has been a growing interest in developing a consensus of testing protocols within the spine community to improve comparison of results between studies. The objective of this study was to perform axial compression experiments on bovine bone-disc-bone specimens at three institutions. No differences were observed between testing environment being air, with PBS soaked gauze, or a PBS bath (P > .206). A 100-fold increase in loading rate resulted in a small (2%) but significant increase in compressive mechanics (P < .017). A 7% difference in compressive stiffness between Labs B and C was eliminated when values were adjusted for test system compliance. Specimens tested at Lab A, however, were found to be stiffer than specimens from Lab B and C. Even after normalizing for disc geometry and adjusting for system compliance, an ∼35% difference was observed between UK based labs (B and C) and the USA based lab (A). Large differences in specimen stiffness may be due to genetic differences between breeds or in agricultural feed and use of growth hormones; highlighting significant challenges in comparing mechanics data across studies. This research provides a standardized test protocol for the comparison of spinal specimens and provides steps towards understanding how location and test set-up may affect biomechanical results.

Transient swelling behavior of the bovine caudal disc.

The intervertebral disc is an avascular composite structure, comprised of the nucleus pulposus (NP) and the annulus fibrosus (AF). Previous tissue-level experiments either examined relative differences in swelling capacity of the two disc regions at a single time point or tested explant structures that did not replicate in situ boundary conditions. Previous joint-level studies that investigated time-dependent fluid flow into the disc provided limited information about swelling-induced intradiscal strains with respect to time and boundary constraints. Therefore, the objective of this study was to investigate time-dependent swelling behavior of the intervertebral disc ex situ. The first study investigated time-dependent free-swelling response of the whole disc and the disc's subcomponents separately (i.e., NP and AF). Findings from this study showed that the swelling rate and swelling capacity of NP explants under free-swelling conditions were greater than AF explants. The second study evaluated the effect of boundary conditions on in-plane strain distributions of intact discs and AF rings. Swelling-induced strain was highly heterogeneous in AF rings, where negative circumferential strains were observed in the inner AF and tensile circumferential strains were observed in the outer AF. Radial strains in AF rings were an order of magnitude greater than circumferential strains. Restricting fluid flow only to the outer AF periphery reduced the swelling of the inner AF. Swelling of intact discs affected both NP and AF swelling behaviors, where NP hydration decreased by 60%. Furthermore, the presence of the NP reduced peak radial strains in the AF and resulted in uniform strain distribution throughout the AF. In conclusion, these studies highlight that tissue hydration and swelling-induced strains largely depend on regional biochemical composition and geometric boundary constraints.