The Micro- and Nano-scale Structure of the Most Superficial Layer of Articular Cartilage

This dissertation illustrates the three-dimensional structure and composition of the most superficial layer of healthy sheep articular cartilage to gain understanding of the role of this layer in the tissue’s function. This was achieved by isolating the layer from the underlying cartilage to study using confocal and atomic force microscopy. The imaging techniques studied the microstructure of the most superficial layer. This knowledge can advance our understanding of the aetiology of osteoarthritis and aid the development of tissue regenerative therapies and diagnostic techniques

History-Dependent Changes to the Structure, Properties, and Function of the Cartilaginous Endplate

The performance of manual lifting is associated with 33-51% of incidental low back injuries in work, leisure, and sport/exercise contexts. To effectively prevent low back injuries and evaluate the risk of occurrence, knowledge on the fundamental pathways of microscopic damage accumulation in lumbar spine tissues is required and lacking in the literature. This thesis broadly explored this knowledge gap in the cartilaginous endplate (CEP), which is a hypothesized origin for compression-induced low back injuries and degenerative changes to the intervertebral disc. Therefore, the global objectives of this dissertation included: 1) to quantify the effect of cyclic compression paradigms on the properties and microstructure of the cartilaginous endplate; 2) to examine the effect of cyclic loading parameters on microscopic and macroscopic injury patterns in the cartilaginous endplate; and 3) to characterize the cycle-dependent ultimate compression trajectories in response to acute loading histories. To address these objectives, in vitro mechanical testing and immunofluorescence staining techniques were developed and performed on intact spinal units and isolated CEP tissue. The effects of joint posture, variation in peak compression force, and loading duration on cycle-dependent changes to spinal joint mechanics, isolated CEP properties, and the pathways of microstructural and constitutive damage were quantified. Compared to neutral joint postures, cyclic loading applied to flexed spinal joints reduced the ultimate strength and CEP stiffness at a given loading duration, irrespective of the peak compression variation. An effect of peak compression variation was observed only within neutral postures and beyond the approximate mid-point of the joint lifespan; a 40% variation reduced the joint strength and CEP stiffness compared to the 10% and 20% variation groups. These altered mechanical properties were supported by evidence of sub-surface microstructural void development followed by damage to native type II collagen proteins within the central CEP region. Data obtained from these in vitro mechanical tests were then used to mathematically characterize the relationships between UCT and loading duration. The second-order polynomial functions demonstrated the depreciation of ultimate compression tolerance and altered safety margins for a given loading history. These data collectively highlighted the importance of spinal joint posture for mediating the damage cascade, which can inform the priorities of job (re)design, clinical intervention, and movement training. The morphology of microstructural injury patterns was also driven by joint posture during sub-threshold cyclic loading, and the lesion size generally progressed as a function of loading duration. That is, cartilage microfractures were more common in neutrally positioned joints, while avulsion and node microinjuries were most common in flexed spinal units. However, on a macroscopic level, the failure morphology was less sensitive to posture and was attributed to the pace of damage accumulation in the sub-chondral bone relative to the hyaline cartilage surface. This notion was experimentally demonstrated by imposing targeted trabecular bone strength deficits within intact vertebrae and performing subsequent fatigue testing. Initially healthy spinal joints resulted in fracture lesions, while spinal joints with pre-existing strength deficits resulted in Schmorl’s nodes over 50% of the time. This dissociation of macroscopic injury mechanisms provided new insights into their prevention, treatment, and diagnosis and ultimately improves the specificity of ergonomics tools that are developed from in vitro experimental data. Overall, this research documents the effects of joint posture, variation in peak compression force, and loading duration on the pathways and time-course of microscopic damage in the cartilage endplate of the spine. These data will be used to broadly inform task design, load management, and injury prevention initiatives in many occupational sectors, specifically public protection (i.e., military, emergency response personnel), health care, manufacturing, and professional/colligate sport where low back injuries are a common and costly cause of personal disability and lost work time. The work of this thesis further advanced the methodology used within the broader field of spine biomechanics and the experimental results represent a significant step to understanding the mechanisms and prevention of lifting-related overuse injuries in the lumbar spine

Experimental and computational studies of the growth plate reserve zone and chondro-osseous junction

The growth plate of a long bone is an organ comprised of a thin layer of hyaline cartilage sandwiched between epiphyseal and metaphyseal bone and surrounded by fibrous tissues. The cartilage tissue can be divided into three histological zones reflecting the activities of the chondrocytes from the epiphysis toward the metaphysis: a reserve, proliferative and hypertrophic zone. Longitudinal growth occurs by a process of endochondral ossification in which cartilage in the hypertrophic zone at the metaphyseal border is calcified and then replaced by bone while new cartilage is produced in the proliferative zone. Application of mechanical loading modulates the chondrocyte activity in the proliferative and hypertrophic zones. As growth continues the growth plate develops into a three-dimensional interlocking interface of hills and valleys, termed mammillary processes, and a layer of compact subchondral bone arises at the border of the reserve zone and epiphysis. The undulations on the metaphyseal side of the growth plate are formed by endochondral ossification. The mechanism by which the undulations between the reserve zone and subchondral epiphysis form has not been elucidated. Recent discoveries of stem-like cells in the reserve zone suggest that reserve zone cells may also modulate growth under mechanical loading. To explore this possible function the present work examined the histology and chemistry of the interface between the reserve zone and epiphyseal bone in a pig model. Elastic and poroelastic multiscale finite element models of the growth plate were developed to investigate the depth-dependent biomechanical microenvironment of reserve zone chondrocytes, particularly cells close to the subchondral bone and proliferative zone. The histological, chemical and computational results suggest that reserve zone chondrocytes near the epiphysis participate in a slower second endochondral ossification front that develops the subchondral bone plate forming undulations that match those on the metaphyseal side. Computational results indicate that dynamic loading engenders fluid shear stresses around reserve zone cells that may signal dividing cells to orient and align in columns. The depth-dependent micro-mechanical environment of the reserve zone cell is highly sensitive to the permeability of the subchondral bone plate and to the rate of loading

Composition-Dependent Mechanisms of Multiscale Tendon Mechanics

Tendons serve as an integral part of the musculoskeletal system by transferring loads from muscle to bone and providing joint mobility and stability. From the physiologically-loading perspective, while progress has been made in evaluating mechanical behavior of different types of tendons in tension, further work is needed to relate tendon mechanics to compositional and microstructural properties, particularly under non-tensile loading modalities (i.e., shear, compression). This information is vital to explore mechanisms of how mechanical signals lead to changes in tendon structure and composition to enable these tissues to function properly, including in in vivo multiaxial loading conditions. From the structural perspective, tendon exhibits a hierarchical organization as collagen is bundled into fibrils, fibers, fascicles, and finally full tissue. Within this hierarchy, linking components are believed to act as connections to maintain mechanical integrity. Three linking components have been proposed, namely elastic fibers, proteoglycans, and collagen crosslinks, however conclusions about their specific mechanical roles, assessed using experimental and computational approaches, are inconsistent. In addition, it remains unknown whether/how these linking components regulate tendon microscale behavior (i.e., at the level of cells) and mechanical signal transfer across length scales. Therefore, this study aimed to (1) develop a protocol that combined a biomechanical test device with two-photon microscopy to measure tendon mechanical strength and multiscale deformation; (2) apply this experimental approach to evaluate region-dependent biomechanics of tendons and related physical mechanisms governing their microscale behavior; (3) determine the role of proteoglycans and elastic fibers in tendon multiscale mechanical behavior using enzyme-treated tendons; and (4) elucidate the contribution of collagen crosslinks to tendon mechanics using in vivo treatment and in vitro culture. We found that different regions of bovine flexor tendon exhibited distinct elasticity, but not viscosity, when subjected to shear and compression, and that fiber sliding and reorganization were the primary modes of microscale deformation. Elastic fibers contributed to supraspinatus tendon (SST) mechanical strength in shear, while proteoglycans appeared to not contribute to SST multiscale biomechanics. Rat SST with decreased collagen crosslink density showed inferior mechanical properties, demonstrating the role of collagen crosslinks on tendon mechanical behavior. Taken together, these results have illustrated tendon composition-mechanics relationships by evaluating mechanical contribution of specific linking components at different length scales. In addition, this work provides insight into mechanical consequences that may accompany extracellular matrix changes during tissue injury/degeneration, and as well provides useful data to aid the design of biomimetic engineered tissues with appropriate structure and composition

Strategies for engineering cartilage with improved content and organization

Cartilage degeneration, from injury or osteoarthritis, is an important problem in current orthopaedic practice. Articular cartilage is unable to repair itself, resulting in a permanent defect and the formation of mechanically inferior fibrocartilage. Tissue engineering is a promising approach for the treatment of cartilage injuries, as it may eventually allow for production of engineered tissue indistinguishable from native cartilage. An important advantage of using tissue engineered material is that you implant a healthy, living tissue. Therefore it is more likely to integrate with the surrounding cartilage tissue. Currently, it is possible to engineer cartilage constructs with native proteoglycan content. However, current tissue-engineered cartilage is not suitable for implantation, because of its insufficient mechanical properties. Two major contributors to this poor mechanical quality are explored in this thesis. First, only 15-35% of the native collagen content is reached in tissue-engineered cartilage. Second, native extracellular matrix (ECM) organization on macro and micro scale is not reproduced. This thesis aims to improve the mechanical quality of tissue-engineered cartilage by exploring approaches to enhance both collagen content and ECM organization. Since our studies are to a great extent dependent on mechano-responsiveness of chondrocytes, we had to establish an appropriate culture model, which then could be used to transmit mechanical forces to the chondrocytes. A well-characterized and widely used model system involves the culturing of chondrocytes in agarose. We demonstrated that loading applied on 3% agarose constructs was sensed and transduced by the embedded chondrocytes. We found that RGD-dependent integrins were involved in mediating compression-induced alterations in ECM gene expression and protein production, and that this effect was dependent on the loading frequency applied. We observed in our and other studies that ECM is deposited mainly in a dense layer close to the chondrocytes. This inhomogeneity is believed to negatively affect mechanical properties of the engineered tissue. The second aim was to improve ECM distribution at the micro scale in chondrocyte-seeded agarose constructs. We demonstrated that distribution of ECM components was more uniform throughout the constructs when these were cultured with no or low agarose concentration, and when cultured in presence of growth factor TGF-ß3. Zonal characteristics in matrix content and distribution have shown to be essential for the mechanical functioning of cartilage tissue, but are not reproduced in tissue-engineered cartilage. Therefore, our next aim was to create depth-dependent zonal variations in engineered cartilage constructs. We explored the hypothesis that depth-dependent mechanical cues, induced by a new, dedicated loading method called ‘sliding indentation’, would stimulate ECM synthesis depth-dependently. Numerical evaluation of this sliding indentation loading regime has shown that it can induce depth-varying strain fields in chondrocyte-seeded agarose constructs. It was confirmed that sliding indentation results in a depth-dependent response by the embedded chondrocytes, which was strongest in the regions that received highest strains. Another shortcoming of current tissue-engineered cartilage is its low collagen fraction. Since the mechanical function of collagen in articular cartilage is to resist tension, we postulated that in order to stimulate collagen formation we need to apply tension to the engineered cartilage constructs. Dynamic tension was applied by the aforementioned sliding indentation loading regime. In two separate studies, we demonstrated that application of dynamic tension induced by sliding indentation stimulated collagen type II production both in periosteum-derived cartilage and in chondrocyte-seeded agarose constructs. In conclusion, it has been shown that application of sliding indentation leads to increased collagen fractions and depth-dependent ECM distribution in tissue-engineered cartilage. The latter is major asset of using sliding indentation over alternative, regular loading protocols for tissue engineering, such as unconfined compression. Furthermore, it was demonstrated that ECM distribution at the micro scale appears more homogeneous in constructs cultured with no or low concentration agarose and in presence of TGF-ß3. These findings may be used to define the optimal culture conditions for tissue engineering of cartilage with native collagen content, depth-dependent matrix organization, and sufficient mechanical properties, which are of pivotal importance for the engineering of mechanically stable, functional tissue engineered cartilage

Biomaterial-Mediated Reprogramming of the Wound Interface to Enhance Meniscal Repair

Endogenous repair of fibrous connective tissues is limited, and there exist few successful strategies to improve healing after injury. As such, new methods that advance repair by enhancing cell migration to the wound interface, extracellular matrix (ECM) production, and tissue integration would represent a marked clinical advance. Using the adult meniscus as a test platform, we hypothesized that ECM density and stiffness increase throughout tissue maturation, and that these age-related changes present biophysical barriers to interstitial cell migration during wound healing. We further posited that modulating the matrix could remove these impediments, enabling endogenous cells to reach the injury site. To test our hypotheses, we compared the microenvironment of fetal and adult meniscal ECM via atomic force microscopy (AFM) indentation and second harmonic generation (SHG) imaging of the collagenous matrix. We also explored interstitial cell mobility through fetal and adult native tissue environments using a three-dimensional ex vivo system. We further investigated strategies that might expedite cell migration, including enzymatic degradation of the ECM with collagenase to reduce matrix stiffness and increase porosity. To restrict these biological manipulations to the wound interface, we fabricated a delivery system in which selected biofactors were stored inside composite electrospun nanofibrous scaffolds and released upon hydration. The ability for bioactive scaffolds to enhance the cellularity and integration of meniscal injuries was evaluated in vivo using tissue explants in a subcutaneous implantation model, as well as an orthotopic meniscal injury model. Our findings suggest that matrix stiffness, density, and organization increase with meniscal development at the expense of cell mobility. Our results also indicate that partial digestion of the wound interface with collagenase improves repair by creating a more compliant and porous microenvironment that facilitates cell migration. Furthermore, when scaffolds containing collagenase-releasing fibers were placed inside meniscal defects, enzymatic digestion was localized and resulted in improved cellular colonization and closure of the wound site, similar to treatment with aqueous collagenase. This innovative approach of targeted delivery may aid the many patients that exhibit meniscal tears by promoting integrative repair, thereby circumventing the pathologic consequences of partial meniscus removal, and may find widespread application in the treatment of injuries to a variety of dense connective tissues

Development and Applications of Advanced Ultrasound Techniques for Characterization and Stimulation of Engineered Tissues

Mechanobiology is central in the development, pathology, and regeneration of musculoskeletal tissues, in which mechanical factors play important roles. Therefore, there is a need for methods to characterize the composition and mechanical properties of developing musculoskeletal tissues over time. Ultrasound elastographic techniques have been developed for noninvasive imaging of spatial heterogeneity in tissue stiffness. However, their application for quantitative assessment of tissue mechanical properties, especially viscoelastic properties, has not been exploited. Additionally, ultrasound energy may be used to apply mechanical stimulation to engineered constructs at the microscale, and thereby to enhance tissue regeneration. We have developed a multimode ultrasound viscoelastography (MUVE) system for assessing microscale mechanical properties of engineered hydrogels. MUVE uses focused ultrasound pulses to apply acoustic radiation force (ARF) to deform samples, while concurrently measuring sample dimensions using coaxial high frequency ultrasound imaging. We used MUVE to perform creep tests on agarose, collagen, and fibrin hydrogels of defined concentrations, as well as to monitor the mechanical properties of cell-seeded constructs over time. Local and bulk viscoelastic properties were extracted from strain-time curves through fitting of relevant constitutive models, showing clear differences between concentrations and materials. In particular, we showed that MUVE is capable of mapping heterogeneity of samples in 3D. Using inclusion of dense agarose microbeads within agarose, collagen and fibrin hydrogels, we determined the spatial resolution of MUVE to be approximately 200 μm in both the lateral and axial directions. Comparison of MUVE to nanoindentation and shear rheometry showed that our ultrasound-based technique was superior in generating consistent, microscale data, particularly for very soft materials. We have also adapted MUVE to generate localized cyclic compression, as a means to mechanically stimulate engineered tissue constructs at the microscale. Selected treatment protocols were shown to enhance the osteogenic differentiation of human mesenchymal stem cells in collagen-fibrin hydrogels. Constructs treated at 1 Hz at an acoustic pressure of 0.7 MPa for 30 minutes per day showed accelerated osteogenesis and increased mineralization by 10 to 30 percent, relative to unstimulated controls. In separate experiments, the ultrasound pulse intensity was increased over time to compensate for changes in matrix properties over time, and a 35 percent increase in mineralization was achieved. We also extended the application of a previously-developed spectral ultrasound imaging (SUSI) technique to an animal model for early detection of heterotopic ossification (HO). The quantitative information on acoustic scatterer size and concentration derived from SUSI was used to differentiate tissue composition in a burn/tenotomy mice model from the control model. Importantly, HO foci were detected as early as one week after injury using SUSI, which is 3-5 weeks earlier than when using conventional micro-computed tomography. Taken together, these results demonstrate that ultrasound-based techniques can non-invasively and quantitatively characterize viscoelastic properties of soft materials in 3D, as well as their composition over time. Ultrasound pulses can also be used to stimulate engineered constructs to promote musculoskeletal tissue formation. MUVE, SUSI, and ultrasound stimulation can be combined into an integrated system to investigate the roles of matrix composition, static mechanical environment, and dynamic mechanical stimuli in tissue regeneration, as well as the interactions of these factors and their evolution over time. Ultrasound-based techniques therefore have promising potential in noninvasively characterizing the composition and biomechanics, as well as providing mechanical intervention in native and engineered tissues as they develop over time.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144116/1/xho_1.pd

Visualisation of Articular Cartilage Microstructure

This thesis developed image processing techniques enabling the detection and segregation of biological three dimensional images into its component features based upon shape and relative size of the features detected. The work used articular cartilage images and separated fibrous components from the cells and background noise. Measurement of individual components and their recombination into a composite image are possible. Developed software was used to analyse the development of hyaline cartilage in developing sheep embryos

Multiphysical modelling of mechanical behaviour of soft tissue : application to prostate

The aim of this thesis is to propose computational methodologies to analyse how the morphological and microstructural changes in the soft tissues, caused by various pathological conditions, influence the mechanical properties of tissue. More importantly, how such understanding could provide more insights into the mechanical properties of tissue for the purpose of quantitative diagnosis. To achieve this objective, statistical analysis of tissue microstructure based on image processing of tissue histology has been carried out. The influence of such microstructural changes due to different pathological conditions has also been compared to the mechanical properties of the tissue by means of the homogenization approach. To understand better the influence of fluid movement in viscoelastic behaviour of tissue, an optimization based method using numerical homogenization that is integrated with fluid-structure interaction (FSI) modelling is presented. The microstructures of soft tissue are treated as bi-phasic materials, solid material representing the cells and extracellular materials and fluid phase for the interstitial fluid. Such proposed method would be beneficial for quantitative assessment of mechanical properties of soft tissue, as well as understanding the role of multiscale microstructural features of soft tissues in its functionality. It is envisaged that this work will pave the road towards more precise characterization of mechanical properties of soft tissue which can be implemented to non-invasive diagnostic techniques, in order to improve the effectiveness of a range of diagnostic methods such as palpation for primary prostate diagnosis and, more importantly, the life quality of patients