Integrin mechanosensing relies on a pivot-clip mechanism to reinforce cell adhesion.

Cells intricately sense mechanical forces from their surroundings, driving biophysical and biochemical activities. This mechanosensing phenomenon occurs at the cell-matrix interface, where mechanical forces resulting from cellular motion, such as migration or matrix stretching, are exchanged through surface receptors, primarily integrins, and their corresponding matrix ligands. A pivotal player in this interaction is the α5ß1 integrin and fibronectin (FN) bond, known for its role in establishing cell adhesion sites for migration. However, upregulation of the α5ß1-FN bond is associated with uncontrolled cell metastasis. This bond operates through catch bond dynamics, wherein the bond lifetime paradoxically increases with greater force. The mechanism sustaining the characteristic catch bond dynamics of α5ß1-FN remains unclear. Leveraging molecular dynamics simulations, our approach unveils a pivot-clip mechanism. Two key binding sites on FN, namely the synergy site and the RGD (Arg-Gly-Asp) motif, act as active points for structural changes in α5ß1 integrin. Conformational adaptations at these sites are induced by a series of hydrogen bond formations and breaks at the synergy site. We disrupt these adaptations through a double mutation on FN, known to reduce cell adhesion. A whole-cell finite-element model is employed to elucidate how the synergy site may promote dynamic α5ß1-FN binding, resisting cell contraction. In summary, our study integrates molecular- and cellular-level modeling to propose that FN's synergy site reinforces cell adhesion through enhanced binding dynamics and a mechanosensitive pivot-clip mechanism. This work sheds light on the interplay between mechanical forces and cell-matrix interactions, contributing to our understanding of cellular behaviors in physiological and pathological contexts.

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.

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.

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.

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.

Direct Quantification of Intervertebral Disc Water Content Using MRI.

BACKGROUND: Water content is a key parameter for simulating tissue swelling and nutrient diffusion. Accurately measuring water content throughout the intervertebral disc (NP = nucleus pulposus; AF = annulus fibrosus) is important for developing patient-specific models. Water content is measured using destructive techniques, Quantitative MRI has been used to estimate water content and detect early degeneration, but it is dependent on scan parameters, concentration of free water molecules, and fiber architecture. PURPOSE: To directly measure disc-tissue water content using quantitative MRI and compare MRI-based measurements with biochemical assays, and to quantify changes in disc geometry due to compression. STUDY TYPE: Basic science, controlled. SPECIMEN: Twenty bone-disc-bone motion segments from skeletally mature bovines. FIELD STRENGTH/SEQUENCE: 7T/3D fast low angle shot (FLASH) pulse sequence and a T2 rapid imaging with refocused echoes (RARE) sequence. ASSESSMENT: Disc volumes, NP and AF volumetric water content, and T2 relaxation times were measured through MRI; NP and AF tissue gravimetric water content, mass density, and glycosaminoglycan content were measured through a biochemical assay. STATISTICAL TESTS: Correlations between MRI-based measurement and biochemical composition were evaluated using Pearson's linear regression. RESULTS: Mechanical dehydration resulted in a decrease in disc volume by up to 20% and a decrease in disc height by up to 35%. Direct water content measurements for the NP was achieved by normalizing MRI-based spin density by NP mass density (1.10 ± 0.03 g/cm3 ). However, the same approach underestimated water content in the AF by ~10%, which may be due to a higher concentration of collagen fibers and bound water molecules. DATA CONCLUSION: Spin density or spin density normalized by mass density to estimate NP and AF water content was more accurate than correlations between water content and relaxation times. Mechanical dehydration decreased disc volume and disc height, and increased maximum cross-sectional area. LEVEL OF EVIDENCE: TECHNICAL EFFICACY STAGE: J. Magn. Reson. Imaging 2020;52:1152-1162.

Historical Review of Combined Experimental and Computational Approaches for Investigating Annulus Fibrosus Mechanics.

Intervertebral disc research has sought to develop a deeper understanding of spine biomechanics, the complex relationship between disc health and back pain, and the mechanisms of spinal injury and repair. To do so, many researchers have focused on characterizing tissue-level properties of the disc, where the roles of tissue subcomponents can be more systematically investigated. Unfortunately, experimental challenges often limit the ability to measure important disc tissue- and subtissue-level behaviors, including fiber-matrix interactions, transient nutrient and electrolyte transport, and damage propagation. Numerous theoretical and numerical modeling frameworks have been introduced to explain, complement, guide, and optimize experimental research efforts. The synergy of experimental and computational work has significantly advanced the field, and these two aspects have continued to develop independently and jointly. Meanwhile, the relationship between experimental and computational work has become increasingly complex and interdependent. This has made it difficult to interpret and compare results between experimental and computational studies, as well as between solely computational studies. This paper seeks to explore issues of model translatability, robustness, and efficient study design, and to propose and motivate potential future directions for experimental, computational, and combined tissue-level investigations of the intervertebral disc.

Relative Nucleus Pulposus Area and Position Alter Disk Joint Mechanics.

Aging and degeneration of the intervertebral disk are noted by changes in tissue composition and geometry, including a decrease in nucleus pulposus (NP) area. The NP centroid is positioned slightly posterior of the disk's centroid, but the effect of NP size and location on disk joint mechanics is not well understood. We evaluated the effect of NP size and centroid location on disk joint mechanics under dual-loading modalities (i.e., compression in combination with axial rotation or bending). A finite element model (FEM) was developed to vary the relative NP area (NP:Disk area ratio range = 0.21-0.60). We also evaluated the effect of NP position by shifting the NP centroid anteriorly and posteriorly. Our results showed that compressive stiffness and average first principal strains increased with NP size. Under axial compression, stresses are distributed from the NP to the annulus, and stresses were redistributed toward the NP with axial rotation. Moreover, peak stresses were greater for disks with a smaller NP area. NP centroid location had a greater impact on intradiscal pressure during flexion and extension, where peak pressures in the posterior annulus under extension was greater for disks with a more posteriorly situated NP. In conclusion, the findings from this study highlight the importance of closely mimicking NP size and location in computational models that aim to understand stress/strain distribution during complex loading and for developing repair strategies that aim to recapitulate the mechanical behavior of healthy disks.

A Novel Method for Repeatable Failure Testing of Annulus Fibrosus.

Tears in the annulus fibrosus (AF) of the intervertebral disk can result in disk herniation and progressive degeneration. Understanding AF failure mechanics is important as research moves toward developing biological repair strategies for herniated disks. Unfortunately, failure mechanics of fiber-reinforced tissues, particularly tissues with fibers oriented off-axis from the applied load, is not well understood, partly due to the high variability in reported mechanical properties and a lack of standard techniques ensuring repeatable failure behavior. Therefore, the objective of this study was to investigate the effectiveness of midlength (ML) notch geometries in producing repeatable and consistent tissue failure within the gauge region of AF mechanical test specimens. Finite element models (FEMs) representing several notch geometries were created to predict the location of bulk tissue failure using a local strain-based criterion. FEM results were validated by experimentally testing a subset of the modeled specimen geometries. Mechanical testing data agreed with model predictions (∼90% agreement), validating the model's predictive power. Two of the modified dog-bone geometries ("half" and "quarter") effectively ensured tissue failure at the ML for specimens oriented along the circumferential-radial and circumferential-axial directions. The variance of measured mechanical properties was significantly lower for notched samples that failed at the ML, suggesting that ML notch geometries result in more consistent and reliable data. In addition, the approach developed in this study provides a framework for evaluating failure properties of other fiber-reinforced tissues, such as tendons and meniscus.

Effect of Hydration on Healthy Intervertebral Disk Mechanical Stiffness.

The intervertebral disk has an excellent swelling capacity to absorb water, which is thought to be largely due to the high proteoglycan composition. Injury, aging, degeneration, and diurnal loading are all noted by a significant decrease in water content and tissue hydration. The objective of this study was to evaluate the effect of hydration, through osmotic loading, on tissue swelling and compressive stiffness of healthy intervertebral disks. The wet weight of nucleus pulposus (NP) and annulus fibrosus (AF) explants following swelling was 50% or greater, demonstrating significant ability to absorb water under all osmotic loading conditions (0.015 M-3.0 M phosphate buffered saline (PBS)). Estimated NP residual strains, calculated from the swelling ratio, were approximately 1.5 × greater than AF residual strains. Compressive stiffness increased with hyperosmotic loading, which is thought to be due to material compaction from osmotic-loading and the nonlinear mechanical behavior. Importantly, this study demonstrated that residual strains and material properties are greatly dependent on osmotic loading. The findings of this study support the notion that swelling properties from osmotic loading will be important for accurately describing the effect of degeneration and injury on disk mechanics. Furthermore, the tissue swelling will be an important consideration for developing biological repair strategies aimed at restoring mechanical behavior toward a healthy disk.

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.

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.

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.

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.

Toward engineering a biological joint replacement.

Osteoarthritis is a major cause of disability and pain for patients in the United States. Treatments for this degenerative disease represent a significant challenge considering the poor regenerative capacity of adult articular cartilage. Tissue-engineering techniques have advanced over the last two decades such that cartilage-like tissue can be cultivated in the laboratory for implantation. Even so, major challenges remain for creating fully functional tissue. This review article overviews some of these challenges, including overcoming limitations in nutrient supply to cartilage, improving in vitro collagen production, improving integration of engineered cartilage with native tissue, and exploring the potential for engineering full articular surface replacements.

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.

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.

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.

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.