Integrating Theoretical and Experimental Methods for Multi-Scale Tissue Engineering of the Annulus Fibrosus of the Intervertebral Disc

There is a critical need for tissue engineered replacements for diseased and degenerated intervertebral discs in order to assuage low back pain while restoring function to the spine. Despite progress by many research groups, it remains a challenge to engineer a replacement tissue that can withstand the complex, demanding loading environment of the spine. Due to the hierarchical organization of the intervertebral disc, successful recapitulation of its functional behavior requires replication of anatomic form and physiologic function over a wide range of length scales. In this work, the technology of electrospinning has been employed for tissue engineering of the annulus fibrosus (AF) of the intervertebral disc using a multi-scale approach. The mechanics of electrospun nanofibrous assemblies was first characterized, focusing on how microscopic organization translates to macroscopic mechanical function. Next, engineered tissues were formed by culturing cells on nanofibrous scaffolds, generating aligned, dense collagenous tissues that replicate the single lamellar organization of the AF. This technology was then expanded to engineer angle-ply laminates that replicated both the anatomic form and mechanical function of the native AF. Finally, these results were further extended to engineer an angle-ply fiber-reinforced hydrogel composite that parallels the macroscopic structural organization of the intervertebral disc. Throughout, mechanical testing and mathematical modeling was used to understand material behavior, quantify functional growth, and guide comparison between engineered AF constructs and native tissue benchmarks. Emphasis has been placed on reconciling compositional and structural observations with their macroscopic mechanical implications, utilizing theoretical models to understand these relationships, and using engineered tissues to improve our understanding of structure-function relations within native fiber-reinforced soft tissues

Multi-scale biomechanical study of transport phenomena in the intervertebral disc

Intervertebral disc (IVD) degeneration is primarily involved in back pain, a morbidity that strongly affects the quality of life of individuals nowadays. Lumbar IVDs undergo stressful mechanical loads while being the largest avascular tissues in our body: Mechanical principles alone cannot unravel the intricate phenomena that occur at the cellular scale which are fundamental for the IVD regeneration. The present work aimed at coupling biomechanical and relevant molecular transport processes for disc cells to provide a mechanobiological finite element framework for a deeper understanding of degenerative processes and the planning of regenerative strategies. Given the importance of fluid flow within the IVD, the influence of poroelastic parameters such as permeabilities and solid-phase stiffness of the IVD subtissues was explored. A continuum porohyperelastic material model was then implemented. The angles of collagen fibers embedded in the annulus fibrosus (AF) were calibrated. The osmotic pressure of the central nucleus pulposus (NP) was also taken into account. In a parallel study of the human vertebral bone, microporomechanics was used together with experimental ultrasonic tests to characterize the stiffness of the solid matrix, and to provide estimates of poroelastic coefficients. Fluid dynamics analyses and microtomographic images were combined to understand the fluid exchanges at the bone-IVD interface. The porohyperelastic model of a lumbar IVD with poroelastic vertebral layers was coupled with a IVD transport model of three solutes - oxygen, lactate and glucose - interrelated to reproduce the glycolytic IVD metabolism. With such coupling it was possible to study the effect of deformations, fluid contents, solid-phase stiffness, permeabilities, pH, cell densities of IVD subtissues and NP osmotic pressure on the solute transport. Moreover, cell death governed by glucose deprivation and lactate accumulation was included to explore the mechanical effect on cell viability. Results showed that the stiffness of the AF had the most remarkable role on the poroelastic behavior of the IVD. The permeability of the thin cartilage endplate and the NP stiffness were also relevant. The porohyperelastic model was shown to reproduce the local AF mechanics, provided the fiber angles were calibrated regionally. Such back-calculation led to absolute values of fibers angles and to a global IVD poromechanical behavior in agreement with experiments in literature. The inclusion of osmotic pressure in the NP also led to stress values under confined compression comparable to those measured in healthy and degenerated NP specimens. For the solid bone matrix, axial and transverse stiffness coefficients found experimentally in the present work agreed with universal mass density-elasticity relationships, and combined with continuum microporomechanics provided poroelastic coefficients for undrained and drained cases. The effective permeability of the vertebral bony endplate calculated with fluid dynamics was highly correlated with the porosity measured in microtomographic images. The coupling of transport and porohyperelastic models revealed a mechanical effect acting under large volume changes and high compliance, favored by healthy rather than degenerated IVD properties. Such effect was attributed to strain-dependent diffusivities and diffusion distances and was shown to be beneficial for IVD cells due to the load-dependent increases of glucose levels. Cell density, NP osmotic pressure and porosity were the most important parameters affecting the coupled mechano-transport of metabolites. This novel study highlights the restoration of both cellular and mechanical factors and has a great potential impact for novel designs of treatments focused on tissue regeneration. It also provides methodological features that could be implemented in clinical image-based tools and improve the multiscale understanding of the human spine mechanobiology

INTEGRATING BIOMECHANICS AND CELL PHYSIOLOGY TO UNDERSTANDING IVD NUTRITION AND CELL HOMEOSTASIS

Back pain associated with degeneration of the intervertebral disc (IVD) is a major public health problem in Western industrialized societies. Degeneration of the IVD changes the osmotic and nutrient environment in the extracellular matrix (ECM) which affects cell behaviors, including: cell proliferation, cell energy metabolism, and matrix synthesis. In addition, a thin layer of hyaline cartilaginous end-plate (CEP) at the superior/inferior disc-vertebral interface was found to play an important role in nutrient supply as well as load distribution in the IVD. Therefore, our general hypothesis is that the CEP regulates the ECM osmotic and nutrient environment which further affects IVD cell energy metabolism and homeostasis. First, based on the triphasic theory, we developed a multiphasic model that considered the IVD tissue as a mixture with four phases: solid phase with fixed charges, interstitial water phase, ion phase with two monovalent species (e.g., Na+ and Cl‾), and an uncharged nutrient solute phase. Our numerical results showed calcification of the CEP significantly reduced the nutrient levels in the human IVD. In cell based therapies for IVD regeneration, excessive amounts of injected cells may cause further deterioration of the nutrient environment in the degenerated disc. To address the lack of experimental data on CEP tissue, the regional biomechanical and biochemical characterization of the bovine CEP was conducted. We found that the lateral endplate was much stiffer than the central endplate and might share a greater portion of loading. Our results also indicated that the CEP could block rapid solute convection and allowed pressurization of the interstitial fluid in response to loading. The energy metabolism properties of human IVD cells in different extracellular nutrient environments were also outlined. We found that human IVD cells prefer a more prevalent glycolytic pathway for energy needs under harsh nutrient environmental conditions and may switch towards oxidative phosphorylation once the glucose and oxygen levels increase. In order to further analyze the effect of the extracellular environment on cell homeostasis, IVD cells were defined as a fluid-filled membrane using mixture theory. The active ion transport process, which imparts momentum to solutes or solvent, was also incorporated in a supply term as it appears in the conservation of linear momentum. Meanwhile, the trans-membrane transport parameters (i.e hydraulic permeability and ion conductance) were experimentally determined from the measurements of passive cell volume response and trans-membrane ion transport using the differential interference contrast (DIC) and patch clamp techniques. This novel single cell model could help to further illuminate the mechanisms affecting IVD cell homeostasis. The objective of this project was to develop a multi-scale analytical model by incorporating experimentally determined IVD tissue and cell properties to predict the ECM environment and further analyzing its effect on cell energy metabolism and homeostasis. This work provided new insights into IVD degeneration mechanisms and cell based IVD regeneration therapies for low back pain

Time-dependent Characterization of Fluid Flow into the Intervertebral Disc

The primary function of the intervertebral disc is to support large, multi-directional loads acting on the spine. The intervertebral disc has a heterogeneous structure, comprised of a gel-like nucleus pulposus (NP) and the annulus fibrosus (AF). The AF has a highly organized structure consisting of collagen fibers oriented in a criss-cross pattern in the alternating layers. Despite differences in composition and structure, water is the primary biochemical constituent of both tissues, accounting for greater than 65% of tissue’s wet weight. The water content of the intervertebral disc fluctuates throughout the day as the magnitude of compressive stress acting on the spine varies with changes in body posture, muscle activity, and external loads. The disc loses water during the day and absorbs water at night when loads are reduced.Due to the avascular nature of the intervertebral disc, cell viability and metabolism rely on the exchange of nutrients and metabolic by-products via diffusion under biochemical gradients and fluid flow modulated by diurnal loading patterns. Hence, investigating fluid flow kinematics under simulated physiological loading conditions is important for understanding healthy disc function and mechanobiology. However, there is a lack of knowledge of fluid flow behavior and recovery mechanics during low loading conditions when disc absorbs water and increases its height. Hence, this dissertation aims to fill in this gap in the literature by evaluating the time-dependent recovery mechanics and fluid flow kinematics of the healthy intervertebral disc during low loading conditions that simulate bed-rest. To achieve this, this study tested bovine bone-disc-bone motion segments under a series of creep and recovery loading conditions. Results showed that time-dependent disc recovery behavior has contributions from both inherent fluid-independent viscoelasticity and fluid-dependent poroelasticity. Intrinsic viscoelastic effects are present at short time scales, providing partial recovery of disc height within minutes of unloading before poroelastic effects come into play. Poroelastic fluid flow dominates recovery at long time scales and is largely driven by the osmotic differential between tissue and its surrounding environment. In vitro biomechanical tests on disc joints monitor changes in disc height to understand the direction, magnitude, and rate of fluid flow through the disc. However, these studies report displacements for the entire bone-disc-bone joint without the ability to identify the region-specific changes during swelling due to fluid flow. To improve our understanding of the complex fluid redistribution within the disc, the second part of this work characterized the time-dependent swelling behavior of the intervertebral disc ex situ. The first experiment monitored time-dependent changes in tissue mass to compare differences in the swelling capacities of the NP and AF explants under free swelling conditions. NP explants experienced a higher swelling rate and equilibrium swelling capacity than AF explants. Specifically, there was a 200% increase in the NP tissue mass and a 70% increase in the AF tissue mass under free swelling conditions. The second experiment used an optical, non-contact measurement method to evaluate the distribution of swelling-induced strains throughout intact discs and AF rings. Axial deformations were fixed to prevent out-of-plane motion during swelling. The first group consisted of AF rings in contact with saline at the outer periphery and the center of the annular ring. The second group included AF rings in contact with saline solution only at the outer periphery. The third group included intact discs in contact with saline at the outer periphery. Tissue swelling due to fluid flow was observed to be a slow process that strongly depends on tissue-specific biochemical properties and physical boundary constraints. For AF rings, negative circumferential strains were observed in the inner AF, while positive circumferential strains were observed in the outer AF. However, restricting fluid flow only to the outer periphery during swelling reduced the swelling capacity of the inner AF. The largest absolute radial strain was observed to be in the outer AF for Group 1 and the outer AF for Group 2. The swelling capacity of the NP was largely reduced when swelling was restricted to occur only in the radial direction or constrained by the surrounding AF. Results from intact discs showed that NP pressurization during swelling reduces peak radial strains in the AF and results in uniform strain distribution throughout the AF. Together these findings provide a better understanding of intervertebral disc mechanics and function, particularly during low loading periods when disc absorbs water and increases its volume due to swelling. In conclusion, fluid flow is a slow, time-dependent process that depends on many factors, including biochemical properties, external osmotic pressure, loading history, and boundary constraints

Micromechanics of the annulus fibrosus: role of biomolecules in mechanical function

Lower back pain, caused by disc degeneration or injury, has a major effect on the United Stated economy, resulting in large medical costs – 2.5% of US health care expenditures (~50 billion dollars) annually [1]. A herniation is a common injury to the intervertebral disc that is characterized as the migration of the inner nucleus pulposus through the layers of the outer annulus fibrosus. There have been many studies quantifying the mechanical characteristics of the annulus fibrosus and modeling the response, both mathematically and computationally. There has been some work investigating the failure mechanisms of the annulus in a degenerative, micromechanical model, however the work for a larger injury model is lacking. Experimental work shows that repetitive, compressive and bending loads of the disc, causing the annulus to fail in tension, will result in catastrophic disc herniation.The goal of this work is to characterize the failure properties of annular lamellae using a micro-mechanical testing protocol with the long-term goal of developing a failure criterion for the annulus fibrosis. Single layered annular samples were obtained from isolated cadaveric lumbar intervertebral discs in one of four orientations: longitudinal, transverse, radial, and circumferential. Uniaxial tensile tests were performed to failure and the engineering constants and failure stresses and strains determined. Key findings showed different of properties between orientations. Failure stress, elastic modulus and Poisson’s ratio were higher when tested in plane to the fibers or lamellae (longitudinal and circumferential) compared to the out-of-plane orientations (transverse and radial) with higher failure strain for out-of-plane than in-plane specimens. This was furthered by a study investigating the role of macromolecules in the intervertebral disc on the micromechanical behavior of the human cadaveric lumbar annulus fibrosus to determine the role these molecules play in annular mechanics.Using composite theory, a model of the annulus fibrosus was used to determine the stresses in each lamella at different loading conditions. Failure envelopes based on the Tsai-Hill criteria were created. The properties were used to create failure envelopes for the annulus which may predict catastrophic failure of the annulus that contribute to disc herniation and lower back pain. Full understanding of the mechanical properties and failure envelopes of the annulus could potentially lead to a failure model for disc tearing and herniation.Ph.D., Mechanical Engineering -- Drexel University, 201

Immuno-modulatory effects of intervertebral disc cells

Low back pain is a highly prevalent, chronic, and costly medical condition predominantly triggered by intervertebral disc degeneration (IDD). IDD is often caused by structural and biochemical changes in intervertebral discs (IVD) that prompt a pathologic shift from an anabolic to catabolic state, affecting extracellular matrix (ECM) production, enzyme generation, cytokine and chemokine production, neurotrophic and angiogenic factor production. The IVD is an immune-privileged organ. However, during degeneration immune cells and inflammatory factors can infiltrate through defects in the cartilage endplate and annulus fibrosus fissures, further accelerating the catabolic environment. Remarkably, though, catabolic ECM disruption also occurs in the absence of immune cell infiltration, largely due to native disc cell production of catabolic enzymes and cytokines. An unbalanced metabolism could be induced by many different factors, including a harsh microenvironment, biomechanical cues, genetics, and infection. The complex, multifactorial nature of IDD brings the challenge of identifying key factors which initiate the degenerative cascade, eventually leading to back pain. These factors are often investigated through methods including animal models, 3D cell culture, bioreactors, and computational models. However, the crosstalk between the IVD, immune system, and shifted metabolism is frequently misconstrued, often with the assumption that the presence of cytokines and chemokines is synonymous to inflammation or an immune response, which is not true for the intact disc. Therefore, this review will tackle immunomodulatory and IVD cell roles in IDD, clarifying the differences between cellular involvements and implications for therapeutic development and assessing models used to explore inflammatory or catabolic IVD environments

Mechanics of biomimetic materials for tissue engineering of the intervertebral disc

Tissue engineering offers a paradigm shift in the treatment of back pain. Engineered intervertebral discs could replace degenerated tissue and overcome the limitations of current treatments that disrupt the biomechanics of the spine. New materials, which exhibit sophisticated mechanical responses, are needed to provide templates for tissue regeneration. These behaviours include time-dependent deformation---facilitating shock absorption and nutrient transfer---and strong material anisotropy and tensile-compressive nonlinearities---providing flexibility in controlled directions. In this work, frameworks for the design of materials with controllable structure-property relationships are developed. The time-dependent mechanical properties of composites of agar, alginate and gelatin hydrogels are investigated. It is shown that the time-dependent responses of the composites can be tuned over a wide range. It is then demonstrated that materials mimicking the fibre-reinforced nature of natural tissues can be developed by infiltrating thick electrospun fibre networks with alginate. These fibre-reinforced hydrogels have tensile and compressive properties that can be separately altered. To better understand the mechanical behaviour of these hydrogel-based materials, improved methods for characterising poroelastic and poroviscoelastic time-dependent material properties using indentation are developed. It is shown that poroviscoelastic relaxation is the product of separate poroelastic and viscoelastic relaxation responses. The techniques developed here provide a methodology to rapidly characterise the properties of time-dependent materials and to create materials with complex structure-property relationships similar to those found in natural tissues; they present a framework for biomimetic materials design. The work in this thesis can be used to inform the design of clinically relevant tissue engineering treatments and help the quarter of a million people each year who undergo spinal surgery to reduce back pain.This PhD was supported by the Cambridge Commonwealth Trus

Hybrid biomaterials with tuneable mechanical property gradients

Sol-gel hybrid materials are made up of covalently bonded and interpenetrating networks of organic and inorganic components and produce a synergy of the properties of those components above the nanoscale. By altering the ratio of inorganic to organic content, the mechanical properties can be tuned. Here, a silica-poly(tetrahydrofuran) hybrid system was developed with the aim to form a graded stiffness structure that could imitate the radial variation in stiffness of the intervertebral disc and address the unmet clinical need of intervertebral disc replacement. Hybrids were formed with a range of silica contents between 4 and 45 wt.%, varying from an elastomeric to a glassy material, with compressive stiffness between 2 and 200 MPa. High compressive strains are recoverable and mechanical properties were maintained on soaking up to 1.5 years and to 10000 cycles in compression. The hybrid surface was shown to support cell attachment and extract solutions containing the hybrid were non-cytotoxic. A novel synthesis method was developed to join hybrid sols during their gelation, forming a single specimen with a variation in silica content along its length, producing a corresponding variation in stiffness. Samples joined in this way were at least as strong as single phase samples in tension and compression. This exploits the gradual gelation process of the hybrid sol, which can also be used to create a successful ink for 3D extrusion printing: porous scaffolds were formed in this way with 27.7 wt.% SiO2. Meniscus and intervertebral disc replacement prototypes were formed and tested under cyclic loading at rates for comparison with human disc data.Open Acces

Rheology

This book contains a wealth of useful information on current rheology research. By covering a broad variety of rheology-related topics, this e-book is addressed to a wide spectrum of academic and applied researchers and scientists but it could also prove useful to industry specialists. The subject areas include, polymer gels, food rheology, drilling fluids and liquid crystals among others