Biomedical Engineering - Theses

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End date: 2021 × Start date: 2020 × Subject: 3D volume correlation ×

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    Mechanical Consequence of Glycosaminoglycan Content and Arrangement in Native Cartilage for Tissue Engineering Applications
    Rathnayake Mudiyanselage, Manula Saubagya Bandara Rathnayake ( 2021)
    Cartilage is a connective tissue found in the body which performs a variety of mechanical and protective functions according to its location. Cartilage is classified into three types: hyaline, elastic, and fibrocartilage. Cartilage related injuries and congenital conditions can have long-term effects on the quality of life of affected individuals. Tissue engineering approaches to repair degenerated cartilage have been investigated since the early 1990s. Producing tissue-engineered cartilage with mechanical properties similar to native cartilage is a key issue reported in literature. The basic building blocks of cartilage include chondrocytes, water, collagen, elastin, proteoglycans, and glycosaminoglycans (GAGs). Proportions of these components vary according to cartilage type. Among those components, the specific GAGs present in each type of cartilage are not well investigated. The structural interactions between GAGs and other extracellular matrix (ECM) macromolecules are overlooked in literature. The differences in mechanobiological environments of different cartilage types are under-reported, and there is a necessity to develop methods that can evaluate the mechanobiological environment of native cartilage, to facilitate better replication of native cartilage in tissue engineering approaches. Taking these gaps into account, the following research aims were formulated. -Explore the glycosaminoglycan mediated interactions between tissue components in different types of cartilage. -Identify the intrinsic glycosaminoglycan expression in native cartilage and investigate how this can be reproduced in tissue-engineered models. -Develop an image-guided micromechanical evaluation (IGME) approach to investigate the mechanobiological environment of the cartilage. Selective GAG depletion was used to explore the variations in GAG mediated interactions in different cartilage. Articular, auricular, meniscal, and nasal cartilage plugs were treated with chondroitinase ABC, guanidine hydrochloride, and hyaluronidase, and remaining GAG content was quantified. The ability of these reagents to deplete GAG depends on molecular and structural interactions between GAGs and other macromolecules in the ECM. GAGs in auricular cartilage showed strong interactions with the rest of the ECM when compared to the other cartilage. Hyaluronidase treatment removed over 99% GAG content from other cartilage but only 76% from auricular, indicating a strong interaction between GAGs in auricular cartilage and ECM or elastin fibres present in the tissue. Overall, this showed GAG-ECM interactions vary according to cartilage type and location, indicating specific structural roles for GAG types present in cartilage. Intrinsic GAG expression of cartilage was then investigated with immunohistochemistry to identify the spatial localisation of different GAG populations in cartilage. An extensive comparison of specific sulphated glycosaminoglycans (sGAG) types (chondroitin sulphate-CS, dermatan sulphate-DS, keratan sulphate-KS, and heparan sulphate-HS) present in high load bearing cartilage (articular and meniscal) and low load bearing cartilage (auricular and nasal) was carried out. Articular and nasal cartilage showed significantly different GAG expression despite being hyaline cartilage. CS and KS were expressed in every cartilage in both extracellular and pericellular areas, but in auricular cartilage middle zone, CS and KS were expressed only in pericellular areas where chondrocytes are bounded by elastin fibres. Expression of DS was expressed only in tensile load-bearing areas of articular cartilage (superficial zone) and meniscal cartilage (outer zone). Cartilaginous regions of other cartilage did not express DS. Differences in GAG expression between low load-bearing cartilage (auricular and nasal) and high load-bearing cartilage (articular and meniscal) indicates that GAG expression may depend on the local mechanobiological environment of cartilage. Immunohistochemistry results showed that GAG in auricular cartilage is associated with elastin fibres. Such spatial interactions were not seen in other cartilage types, which has collagen as the main fibrous component. Therefore, the effect of ECM components on intrinsic GAG production was investigated in tissue engineering models. Bovine articular chondrocytes were encapsulated in three groups of hydrogel beads containing 1) alginate, 2) alginate and collagen, 3) alginate, collagen, and elastin. Then CS, DS, and KS expression were investigated with immunohistochemistry at day 0, 7, 21, and 35 of culture. Expression and distribution of GAG types were compared between the different groups. CS was more concentrated in pericellular areas. With time, CS staining spread over a wider area in the beads with collagen and elastin. DS showed a more uniform distribution in the matrix. DS staining intensity in beads with collagen and elastin was higher when compared to alginate only beads. KS was only expressed by few cells. Results indicate that collagen and elastin have an effect on distribution patterns of GAG in 3D in vitro environments. Previous studies showed the GAG expression and their interactions in cartilage could depend on the local mechanobiological environment. Therefore, to visualise the mechanics involved in the local mechanobiological environment a novel image-guided micromechanical evaluation (IGME) technique capable of evaluating the local collagen and elastin deformation in cartilage was developed. Mechanical compression, multiphoton imaging, and digital volume correlation (DVC) were combined to analyse the displacement fields of collagen and elastin in auricular cartilage. Preliminary results suggest the feasibility of this method to analyse soft tissue mechanical deformation in 3D space. A custom-built compression device that can be placed under an objective of a multiphoton microscope was used to image cartilage under different strain levels (5%, 10%, 15%, and 20%). An image preprocessing pipeline was developed to segment collagen and elastin from the multiphoton images. 3D images were then used to perform DVC with TomoWarp2 software. This is the first study to evaluate the feasibility of analysing collagen and elastin deformation in cartilage without using markers. This method also allows assessment of individual components without altering the tissue composition. Therefore, this approach can be used to investigate mechanics in cartilage mechanobiological environment without altering it. In this work, it was shown that GAG-ECM interactions are unique to both the cartilage type and anatomical location. This variation of GAG also suggests the presence of different GAG populations in different cartilage. Therefore, spatial localisation of different GAG populations in different articular, auricular, meniscal, and nasal cartilage were investigated. GAGs present in auricular cartilage showed pericellular localisation pattern associated with elastin fibres present. This specific localisation pattern was not seen in other cartilage which had collagen as the main fibrous component. Therefore, effect of collagen and elastin on the production of different GAG types was investigated in 3D in vitro environments. In vitro culture of chondrocytes showed that GAG dispersion patterns are significantly different from the native GAG dispersion patterns. Finally, an IGME technique that would facilitate observing of the behaviour of individual ECM components in cartilage mechanobiological environment was developed and feasibility of that method was assessed.