Dissertations / Theses on the topic 'Cartilage structure'

En el disseny de dispositius mèdics existeixen diversos casos en els quals és necessària la utilització de superfícies bioactives per aconseguir la integració òptima d’un implant amb el teixit que l’envolta. L’enginyeria de superfícies planteja diferents solucions, tot i així, per algunes aplicacions, l’obtenció d’una unió íntima entre el teixit i l’implant encara és un repte clínic. En aquest treball, presentem una tècnica que permet obtenir superfícies biomimètiques en qualsevol substrat que pugui ser sotmès a modificació per plasma. Com a proba de concepte, hem aplicat la tecnologia desenvolupada en l’obtenció d’un scaffold heterogeni per la regeneració del teixit osteocondral, amb un gran potencial per ser utilitzat com a teràpia regenerativa. Un dels grans reptes en la regeneració osteocondral, és assolir un grau elevat de semblança amb l’estructura articular, des de l’òs subcondral fins a la superfície articular. La nostra metodologia permet la immobilització d’un hidrogel que imita el teixit cartilaginós a la superfície d’una plataforma bioceràmica, la qual reprodueix el teixit ossi. Aquesta última, actuarà com a suport mecànic i punt d’ancoratge a l’òs subcondral, a la vegada que proporcionarà un reservori de ions de calci i de fosfat que ajudaran a la creació del gradient de duresa present en les articulacions. Així doncs, en aquesta tesi hem treballat en el disseny de les diferents parts que conformaran el scaffold. En primer lloc, per tal d’aprofundir en la creació del gradient de duresa, hem estudiat la bioactivitat de diferents substituts ossis bioceràmics comercials, els quals son candidats potencials per ser utilitzats en la construcció del scaffold. A continuació, hem validat la viabilitat del recobriment polimèric obtingut per PECVD en substrats bioceràmics i hem demostrat que no compromet la seva bioactivitat. A més, hem demostrat que la modificació superficial permet l’obtenció d’una interfície estable, que no es veu alterada per canvis fisiològics i permet l’autoensamblatge de l’hidrogel. Els estudis in vitro realitzats demostren que la tecnologia d’immobilització preserva la viabilitat cel·lular, i que la formulació permet la migració cel·lular a més de proporcionar un entorn adequat per la diferenciació condrogènica i osteogènica de cèl·lules mare mesenquimals.
En el diseño de dispositivos médicos existen numerosos casos en los que es necesaria la utilización de superficies bioactivas para lograr la integración óptima de un implante con el tejido que le rodea. La ingeniería de superficies propone diferentes soluciones, sin embargo, en determinadas aplicaciones, la obtención de una unión íntima entre el tejido y el implante aún es un reto clínico. En el presente trabajo, presentamos una técnica que permite la obtención de superficies biomiméticas en cualquier sustrato que pueda ser sometido a modificación por plasma. Como prueba de concepto, hemos aplicado la tecnología desarrollada en la obtención de un scaffold heterogéneo para la regeneración del tejido osteocondral, con un gran potencial para ser usado como terapia regenerativa. Uno de los grandes retos en la regeneración osteocondral, es lograr un grado elevado de semejanza con la estructura articular, desde el hueso subcondral hasta la superficie articular. Nuestra metodología permite la inmovilización de un hidrogel que imita el tejido cartilaginoso en la superficie de una plataforma bioceràmica, la cual reproduce el hueso. Ésta última, actuará como soporte mecánico y punto de anclaje al hueso subcondral, a la vez que proporcionará un reservorio de iones de calcio y fosfato que ayudarán en la creación del gradiente de dureza presente en las articulaciones. Así pues, en esta tesis hemos trabajado en el diseño de las diferentes partes que conformaran el scaffold. En primer lugar, para profundizar en la creación del gradiente de dureza, hemos estudiado la bioactividad de diferentes sustitutos óseos biocerámicos comerciales, los cuales son candidatos potenciales para ser utilizados en la construcción del scaffold. A continuación, hemos validado la viabilidad del recubrimiento polimérico obtenido por PECVD en sustratos biocerámicos y hemos demostrado como no compromete su bioactividad. Además, hemos demostrado como la modificación superficial permite la obtención de una interfaz estable, que no se altera por cambios fisiológicos, la cual permite el autoensamblaje del hidrogel. Los estudios in vitro realizados demuestran que la tecnología de inmovilización preserva la viabilidad celular, y que la formulación permite la migración celular además de proporcionar un entorno adecuado para la diferenciación condrogénica y osteogénica de células madre mesenquimales.
In medical device engineering, there are several cases where there is an imperative need of obtaining bioresponsive surfaces to achieve an optimal integration of a certain biomaterial with the surrounding tissue. Surface engineering has provided different approaches, however for certain applications obtaining an intimate bonding between the tissue and the implant remains a clinical challenge. In this work, we present a newly developed technique that allows the obtention of biomimetic surfaces onto any substrate that can be subject to plasma modification. As a proof of concept, we have applied the technology to obtain a heterogeneous scaffold for osteochondral repair, which has a great potential to be used as regenerative therapy. One of the great challenges in osteochondral repair is achieving a high degree of mimicry of the whole joint structure, from the subchondral bone to the surface of hyaline cartilage. Our methodology allows the immobilization of a cartilage-like hydrogel onto a bone-like bioceramic platform by means of a polymeric coating. The bioceramic acts not only as mechanical support and anchoring point to the subchondral bone, but also it acts as a reservoir of calcium and phosphate ions, which through diffusion help in the creation of the stiffness gradient present in joints. Thus, in the present thesis, we have worked on the design of the different parts that will form the osteochondral heterogeneous scaffold. First, to gain insight into the stiffness gradient creation, we have studied the bioactivity of different commercially available bioceramic bone substitutes, which are potential candidates to be used as bone-like platform. Next, we have validated the viability of the polymeric coating obtained through PECVD in this type of biomaterials and shown how it does not compromise their bioactive properties. Moreover, we have demonstrated how the designed surface modification allows the obtention of a stable interface, which is not disrupted by physiological changes, that enables the subsequent self-assembly of a cartilage-like hydrogel. In vitro studies show how our immobilizing technology preserves cell viability, and that our hydrogel formulation enables cell migration as well as it provides a suitable environment for both chondrogenic and osteogenic differentiation of mesenchymal stem cells.

The chondroitin sulfate proteoglycan (CSPG) aggrecan forms link protein-stabilised complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other CSPGs (i.e., versican, brevican and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this thesis, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells, purified to homogeneity and functionally characterised. The recombinant human proteins were found to have properties similar to those described for the native molecules. For example cLP formed dimers, and HA decasaccharides (HA 10-mers) were the minimum size that could compete effectively for their binding to polymeric HA. In addition, gel filtration and protein cross-linking/MALDI-TOF peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other hi solution yet formed ternary complexes with HA24. N-linked glycosylation of VG1 and AG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Additionally, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences hi the conformation of HA stabilised on binding these proteins. To further investigate protein-HA interactions, fluorescent HA oligosaccharides were prepared and characterised. HA oligosaccharides labelled with the fluorophore 2-aminobenzoic acid (2AA) from four to 40 residues hi length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterisation of HA oligosaccharides was facilitated by 2AA derivitisation since it enhanced signals in MALDI-TOF mass spectrometry and improves fluorophore-assisted carbohydrate electrophoresis (FACE) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC) labelled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatised oligosaccharides, as demonstrated by their ability to compete for polymeric hyaluronan binding to VG1, AG1 and cLP. An electrophoretic mobility shift assay was used to study VG1 binding to 2AA-labelled HA 8-, 10-, 20-, 30- and 40-mers and although no stable VG1 binding was observed to labelled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA 10-mers was estimated from densitometry analysis of the free oligosaccharide. Interactions involving 2AA labelled HA 20-, 30-, and 40-mers with VG1 also displayed positive cooperativity. Therefore, oligosaccharides labelled with 2-aminobenzoic acid are biologically active and show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

La presence de fibronectine a ete demontree par des methodes biochimiques et immunohistochimiques dans le cartilage arthrosique et les liquides synoviaux. La tenascine et les isoformes ed-a and ed-b de la fibronectine sont presentes dans le cartilage pathologique mais absentes du cartilage normal. Le taux plasmatique de la fibronectine ed-a est augmente dans la polyarthrite rhumatoide et est liee a la presence d'une vascularite et/ou d'un syndrome de sjogren. L'accumulation de la fibronectine dans le cartilage arthrosique est due a une augmentation de la biosynthese de cette glycoproteine qui est particulierement prononcee dans la zone cartilage adjacente a la zone de denudation de l'os chondral. La fibronectine apparait fragmentee dans les extraits de cartilage arthrosique, les milieux de culture de chondrocyte et les liquides synoviaux. L'importance de la fragmentation de la fibronectine dans les liquides synoviaux de la polyarthrite rhumatoide est due a une augmentation de l'activite des enzymes localement presentes. L'elastase leucocytaire ne semble pas etre responsable de la degradation de la fibronectine synoviale. Parmi les fragments de la fibronectine, il existe au sein du liquide synovial, une correlation entre les fragments de 130 kda, 70 kda et les activites de type gelatinase. Les glycoproteines de structure pourraient jouer un role dans les processus de reparation du cartilage

Incremental marks formed in the vertebral cartilage of most sharks, skates and rays are widely used as indicators of age in elasmobranch growth studies. Such information is essential for fisheries management, thus vertebral ageing has become an invaluable tool for investigating elasmobranch life history. Unfortunately, lack of information on the processes that regulate vertebral growth and mineralization limit efforts to correlate episodic stimuli with increment production. to address this research need, this dissertation investigated these processes through a detailed study on the vertebral cartilage of the clearnose skate, Raja eglanteria. Histologic observations indicated that changes in appositional cartilage growth are the catalyst for increment formation. Observations by scanning electron microscopy supported this conclusion by demonstrating that growth increments differ in the proportion of cells to mineralized matrix. In contrast, elemental analysis of vertebrae using energy-dispersive spectrophotometry demonstrated no change in mineral concentration between seasonal growth layers. This observation was advantageous, because uptake of the radiotracer &\sp{lcub}45{rcub}&Ca was ineffective in estimating the rates of vertebral calcification in captive R. eglanteria. Failure of this standard method appeared to reflect the free exchange of calcium between skeletal and serologic reservoirs. An in vitro method for measuring vertebral growth was developed using incorporation of &\sp{lcub}35{rcub}&S-sulfate as a marker for cartilage matrix synthesis. Certain conserved among elasmobranchs and higher vertebrates. The growth hormone-dependent serum factor insulin-like growth factor-I (IGF-I) increased vertebral matrix synthesis, suggesting an important role for this hormone in regulating elasmobranch skeletal growth. In contrast, corticosterone reduced &\sp{lcub}35{rcub}&S-sulfate uptake in vertebral cartilage, suggesting an inhibitory role for glucocorticosteroids in elasmobranch chondrogenesis. Calcitonin also inhibited vertebral matrix synthesis and, in vivo, may play some role in skeletal development or mineral homeostasis. Finally, nutritional status also appeared to influence vertebral growth in vitro, perhaps indirectly through effects on hormone production. In sum, this dissertation identified changes in cartilage growth as the impetus for vertebral growth zone production in R. eglanteria. Physiological mechanisms that likely regulate vertebral growth are described in this pioneer study on elasmobranch skeletal growth.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.
Includes bibliographical references.
Chondroitin sulfate is a critical component of articular cartilage due to its contribution to the tissue's resistance to compressive deformation. Alterations in the biosynthesis of this molecule over time could impact the ability of the tissue to perform its necessary functions. Several factors have been shown to alter the biosynthesis of chondroitin sulfate in cartilage; among them are age, disease, depth of tissue, and mechanical compression. Specifically, mechanical compression has been shown to have a significant effect on the sulfation pattern and chain length and number in cartilage explant studies. The mechanisms that govern these alterations, however, have not been determined. The purpose of this study is to examine the effects of mechanical compression on chondroitin sulfate biosynthesis and analyze the roles of two possible mechanisms; enzyme transcription and organelle deformation. The effects of mechanical compression on the transcription rates of enzymes associated with the biosynthesis of chondroitin sulfate have not been previously studied. To perform this study in a bovine model, portions of the bovine genome had to be sequenced, PCR primers designed, and bulk expression levels determined. Static compression resulted in the significant up-regulation of two genes of interest: chondroitin sulfate and GalNAc 4S,6-sulfotransferase.
(cont.) Dynamic compression resulted in the significant up-regulation of the three sulfotransferases responsible for the bulk of sulfation in cartilage tissue. These results indicate a transient mechanotransduction reaction that differs based on the load regime. The effect of mechanical loading on the biosynthesis of chondroitin sulfate has been studied previously, however, this study seeks to examine more comprehensive loading regimes. Static compression and release resulted in an increase in 6-sulfation and a decrease in 4-sulfation that lasted to 48 hours after release of compression. Dynamic compression and release had the opposite effect on sulfation ratio, with an increase in 4-sulfation compared to 6-sulfation. The transcription changes seen in this study do not indicate the changes that occur in the end products of synthesis. Other factors may play a larger role, such as precursor availability or transport through the Golgi apparatus. Intracellular organelles react to static compression of the surrounding tissue in one of two manners. The majority of organelles deform much as the nucleus, proportionally in volume and shape to the cell. The Golgi apparatus appears to retain a significant portion of its volume relative to the cell and other organelles. In addition, it reforms structurally into a highly ordered stacked appearance.
(cont.) Osmotic forces within the Golgi may allow it to balance the osmotic load in the cytoplasm and resist compression and altered trafficking of the Golgi may in turn produce the altered appearance. Recent microscopy experiments on the Golgi apparatus utilizing two-photon microscopy have allowed us to examine the reaction of live tissue to static compression. These results illustrate the significant, but differing, effects of static and dynamic compression on the biosynthesis of chondroitin sulfate. The effects of these compression types on the transcription of enzymes responsible for this biosynthesis cannot fully explain the changes seen in newly synthesized chondroitin sulfate. Organelle reorganization has been shown to occur in response to static load and it is possible that altered organelle trafficking plays a role in this altered biosynthesis. Further studies are necessary to determine the final effect of the altered transcription and organelle structure on the manufacture of this important cartilage molecule.
by Jon D. Szafranski.
Ph.D.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Levenston, Marc; Committee Member: Garcia, Andres; Committee Member: Koros, William; Committee Member: Sambanis, Athanassios; Committee Member: Temenoff, Johnna; Committee Member: Vidakovic, Brani.

Les hydrogels à base d’un dérivé cellulosique silanisé : hydroxypropylméthylcellulose silanisé (HPMC-Si) ont été développés en tant que biomatériaux pour une application en ingénierie tissulaire du cartilage articulaire. La dérivatisation inorganique par une fonction siloxane d’un polysaccharide permet d’obtenir un gel injectable polycondensant in situ. L’objectif de cette thèse était de disperser des nanofibres de silice (NFs) au sein de ces hydrogels afin d’améliorer leurs caractéristiques rhéologiques et mécaniques. A 3 wt% de NFs, les hydrogels nanocomposites possèdent un module en compression 5 fois supérieur à celui de l’hydrogel pur et restent cytocompatibles avec des chondrocytes et des cellules souches. De manière parallèle, un travail sur la compréhension de la structure du polysaccharide silanisé lui-même ainsi que sur la description de la structure, la morphologie et la dynamique de l’eau dans les hydrogels d’HPMC-Si a été réalisé. Il a été déterminé que l’eau confinée dans les hydrogels d’HPMC-Si est présente sous deux formes : l’eau d’hydratation et l’eau dite « bulk ». L’eau d’hydratation est en interaction avec les parties hydrophiles du polymère via les liaisons hydrogène, tandis que l’eau « bulk » se comporte comme de l’eau volumique classique. Les mesures des coefficients de diffusion montrent qu’à 300 K, les molécules d’eau « bulk » diffusent sur les distances de l’ordre de 10 μm sans être affectées par la présence du réseau polymère. Nous pensons donc que les hydrogels d’HPMC-Si ont une morphologie hiérarchisée, avec des pores de taille micrométrique dont les parois sont constituées de maillage nanométrique d’HPMC-Si
The hydrogels based on a cellulosic derivative, silanized hydroxypropyl methylcellulose (Si-HPMC), are developed as biomaterials for applications in articular cartilage tissue engineering. The inorganic derivatization of a polysaccharide utilizing a silane function provides an injectable gel which can polycondense in situ. Moreover, dispersion of silica nanofibers within the hydrogels enhances their rheological and mechanical properties. With 3 wt% of nanofibers the nanocomposite hydrogel’s compressive modulus is 5 times higher than that of the pure hydrogel. Such a nanocomposite hydrogel remains cytocompatible with respect to chondrocytes and human stem cells. At the same time, a study on silanized polysaccharide structure as well as a description of the structure and morphology of the gel, then a study of the water dynamics in the Si-HPMC hydrogels are realised. The water confined in Si-HPMC hydrogels is found to exist in two different forms: water of hydration and « bulk » water. The water of hydration interacts with the hydrophilic parts of the polymer by hydrogen bond formation, while the « bulk » water behaves as ordinary bulk water. The measurements of diffusion coefficient at 300 K show that the molecules of « bulk » water diffuse over distances in the order of 10 μm without being affected by the polymer network presence. Thus, the Si-HPMC hydrogels seem to have a hierarchically organized morphology with micrometer sized pores whose pore walls are made of a nanometric mesh

Abstract Osteoarthritis (OA) is a heterogenic joint disease significantly affecting the quality of life of a patient, causing pain and disability. OA causes degenerative changes to the structure and composition of articular cartilage and subchondral bone. Currently, effective treatments for OA are limited, partly due to limitations in defining the imaging biomarkers of early OA. Improvement of imaging modalities in OA research and clinical setup is a requirement for quantitating early OA-related tissue features. In the clinical and preclinical setup, computed tomography (CT) enables imaging of bone and, using specific contrast agents, articular cartilage. The aim of this study is to create and validate novel micro-computed tomography (μCT) methods to quantify OA-related features and modifications in articular cartilage and subchondral bone. Contrast-enhanced μCT methods for imaging the collagen (phosphotungstic acid (PTA) and phosphomolybdic acid (PMA)) and GAG (CA4+) content of the articular cartilage in vitro were validated against various reference methods measuring the biochemical composition of articular cartilage. To improve the μCT imaging of subchondral bone, grey-level co-occurrence matrix (GLCM) based analysis of sub-resolution features of subchondral bone was introduced. In addition, to test the translatability of the GLCM-based analysis to clinical resolution, sub-resolution features extracted from clinical cone-beam CT were validated against the subchondral bone morphometrics from the μCT. PTA showed stronger association with the collagen content of the articular cartilage compared to PMA. PTA was also associated with collagen content even in degraded articular cartilage. CA4+ distribution was found to accumulate in chondrons and surrounding areas, suggesting that it is a prominent contrast agent for high-resolution μCT studies of chondrocytes. The GLCM-based analysis of subchondral bone provided information on cellular structure from μCT images and trabecular bone micro-structures from clinical CT images. In conclusion, μCT imaging can provide quantitative information on the collagen content and chondrons of articular cartilage, as well as on osteocytes in subchondral bone. The methods presented here extend the tools for researchers to quantify osteochondral tissue modifications in OA. Furthermore, the developed image processing tools could be translatable to the clinical CT
Tiivistelmä Nivelrikko on heterogeeninen niveltauti, joka huonontaa yksilön elämän laatua aiheuttaen kipua ja liikuntakyvyttömyyttä. Nivelrikko aiheuttaa nivelkudosten rappeumaa vaikuttaen mm. ruston ja rustonalaisen luun rakenteeseen ja koostumukseen. Nivelrikon kudosmuutosten kuvantamisen kehittäminen ja määrällinen tutkiminen taudin alkuvaiheissa tukisivat nykyisten nivelrikon hoitomenetelmien kehittämistä. Kliinisessä käytössä ja perustutkimuksessa, tietokonetomografia (TT) mahdollistaa luukuvantamisen ja varjoaineita käytettäessä myös rustokuvantamisen. Tämän väitöskirjan tavoitteena on esitellä ja validoida uusia mikrotietokonetomografia-menetelmiä (μTT) nivelrikon rusto- ja luumuutosten määrälliseen tutkimukseen. Varjoaineavusteisia μTT in vitro menetelmiä ruston kollageenin (fosfovolframihappoa (PTA) ja fosfomolybdeenihappoa (PMA)) ja GAG (CA4+) jakauman määrälliseen tutkimukseen validoitiin useilla eri ruston biokemiallista koostumusta mittaavilla vertailumenetelmillä. Rustonalaisen luun kuvantamista kehitettiin soveltamalla harmaasävyjen tekstuurianalyysiä, jolla pyrittiin tunnistamaan kuva-alkiota pienempiä luurakenteita. Rustonalaisen luun μTT-kuvien analyysien tulokset validoitiin synkrotronisäteilyyn perustuvan μTT:n avulla. Lisäksi tekstuurianalyysin soveltuvuutta testattiin kliinisen resoluution kartiokeilan TT-kuville. Kuvista analysoituja tekstuuriparametrejä verrattiin μTT:lla mitattuun todelliseen rustonalaisen luun rakenteeseen. Väitöskirjan tulokset osoittavat, että PTA on spesifimpi kollageenille testatuista varjoaineista ja sen jakauma on verrannollinen kollageenijakaumaan jopa rappeutuneessa nivelrustossa. GAG-spesifisen varjoaineen CA4+:n todettiin kerääntyvän myös kondroneihin, mikä viittaa siihen, että kyseinen varjoaine soveltuisi potentiaalisesti rustosolujen korkean resoluution μTT-tutkimuksiin. Rustonalaisen luun μTT-kuvista analysoitujen tekstuuriparametrien havaittiin olevan verrannollisia osteosyyttien tilavuusfraktion kanssa. Väitöskirjassa esitettyjen tulosten perusteella μTT-kuvantaminen tarjoaa kvantitatiivisen menetelmän nivelruston kollageenijakauman ja rustosolujen sekä rustonalaisen luun osteosyyttien tutkimuksiin. Väitöskirjassa esitetyt menetelmät laajentavat jo olemassa olevaa tutkimusmenetelmien kirjoa nivelrikon aiheuttamien nivelrusto- ja luumuutosten tutkimuksessa. Lisäksi kehitetyt kuva-analyysimenetelmät voivat tarjota tarkempaa tietoa kliinisestä TT:sta

Abstract Osteoarthritis (OA) is the commonest joint disease in the world, and it has a major socioeconomic impact. OA causes progressive degenerative changes in the composition and structure of articular cartilage and subchondral bone. Clinical diagnosis of OA is based on physical examination and qualitative evaluation of changes on plain radiographs. Current clinical imaging methods are subjective or insensitive to early OA changes. Therefore, new methods are needed so as to quantify composition of the cartilage and characteristics of the subchondral bone. The aim of this thesis was to evaluate the potential of clinically applicable X-ray-based methods for the assessment of the cartilage proteoglycan content as well as the structure and density of subchondral bone in a knee joint. Subchondral bone density and structure (local binary patterns, Laplacian, and fractal-based algorithms) analysis methods for two-dimensional (2-D) plain radiographs were validated against three-dimensional (3-D) bone microarchitecture obtained from micro-computed tomography ex vivo and applied to plain radiographs in vivo. Furthermore, a method developed for the evaluation of articular cartilage proteoglycan content from computed tomography (CT) was validated against a delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC), which is widely used as a proteoglycan sensitive method, in subjects referred for an arthroscopy of the knee joint. Subchondral bone density and structure evaluated from 2-D radiographs were significantly related to the bone volume fraction and true 3-D microarchitecture of bone, respectively. In addition, bone density- and structure-related parameters from radiographs were significantly different among subjects with different stages of OA. Cartilage proteoglycan content evaluated from CT was significantly related to dGEMRIC method. Furthermore, dGEMRIC was associated with bone structure from a 2-D radiograph. In conclusion, analysis of bone structure and density is feasible from clinically available 2-D radiographs. A novel CT method sensitive to proteoglycan content should be considered when a 3-D view of cartilage quality is needed
Tiivistelmä Nivelrikko on maailman yleisin nivelsairaus. Se aiheuttaa merkittävää kärsimystä potilaille, ja sillä on suuri taloudellinen vaikutus yhteiskuntaan. Nivelrikko aiheuttaa palautumattomia muutoksia nivelrustokudoksen ja rustonalaisen luun koostumukseen ja rakenteeseen. Nivelrikon diagnoosi perustuu kliiniseen tutkimukseen ja röntgenkuvien silmämääräiseen arviointiin. Nykyiset nivelrikon kliiniset kuvantamismenetelmät ovat subjektiivisia eivätkä riittävän tarkkoja nivelrikon varhaisten muutosten osoittamiseen, minkä vuoksi rustokudoksen koostumuksen ja rustonalaisen luun muutosten arviointiin tarvitaan uusia menetelmiä. Tämän väitöskirjantyön tarkoituksena oli tutkia uusien röntgensäteilyyn perustuvien menetelmien soveltuvuutta polvinivelen rustokudoksen proteoglykaanipitoisuuden sekä luun tiheyden ja rakenteen arviointiin. Rustonalaisen luun tiheyttä ja rakennetta arvioitiin digitaalisesta röntgenkuvasta tietokonepohjaisilla menetelmillä ja tuloksia verrattiin mikrotietokonetomografiassa nähtävään luun kolmiulotteiseen rakenteeseen. Röntgenkuvasta laskettavia muuttujia verrattiin myös eriasteisesta nivelrikosta kärsivien henkilöiden välillä. Rustokudoksen proteoglykaanipitoisuutta epäsuorasti mittaavaa tietokonetomografiamenetelmää verrattiin vastaavaan magneettikuvausmenetelmään henkilöillä, jotka olivat menossa polven niveltähystykseen. Röntgenkuvasta laskettu rustonalaisen luun tiheys ja rakenne olivat tilastollisesti selkeästi yhteydessä luun tilavuusmäärään ja mikrorakenteeseen, ja ne erosivat eriasteisesta nivelrikosta kärsivien henkilöiden välillä. Proteoglykaanipitoisuutta arvioivien tietokonetomografia- ja magneettikuvausmenetelmien välillä oli tilastollisesti merkitsevä korrelaatio. Ruston proteoglykaanipitoisuutta arvioivan magneettikuvausmenetelmän ja röntgenkuvasta laskettavan luun rakenteen välillä oli myös tilastollinen yhteys. Loppupäätelmänä voidaan todeta, että luun tiheyttä ja rakennetta on mahdollista arvioida kliinisesti saatavilla olevista röntgenkuvista. Tietokonetomografiamenetelmän käyttöä tulee harkita tutkimuksissa silloin, kun rustokudoksen tilasta halutaan kolmiulotteista tietoa

Traumatic injuries lead to articular cartilage lesion formation and result in the development of osteoarthritis. Recent research suggests that the early stage of mechanical injuries involve cell death (apoptosis and necrosis) and inflammation. In this thesis, we focus on building mathematical models to investigate the biological mechanism involving chondrocyte death and inflammatory responses in the process of cartilage degeneration. Chapter 1 describes the structure of articular cartilage, the process of carti- lage degeneration, and reviews of existing mathematical models. Chapter 2 presents a delay-diffusion-reaction model of cartilage lesion formation under cyclic loading. Computational methods were used to simulate the impact of varying loading stresses and erythropoietin levels. The model is parameterized with experimental results, and is therefore clinically relevant. Due to numerical limitations using delay differential equations, a new model is presented using tools for population dynamics. Chapter 3 presents an age and space-structured model of articular cartilage lesion formation un- der a single blunt impact. Age structure is introduced to represent the time delay in cytokine synthesis and cell transition. Numerical simulations produce similar tempo- ral and spatial patterns to our experimental data. In chapter 4, we extend our model under the cyclic loading setting. Chapter 5 builds a spatio-temporal model adapted from the former models, and investigates the distribution of model parameters using experimental data and statistical methods. Chapter 6 concludes.

Proteoglycan 4 (PRG4) is a mucinous proteoglycan with an apparent molecular weight of -345 kDa. It has been detected in a variety of tissues including cartilage, tendon, bone heart and liver, and is also known as cartilage superficial zone protein (SZP), lubricin, megakaryocyte stimulating factor (MSF) precursor protein and camptodactyly-arthropathy-coxa vara pericarditis (CACP) protein. PRG4 has been shown to reduce friction in joints, and immunological studies have located PRG4 on the surface of articular cartilage, in synovial fluid, on the surface of meniscus and on the surface of mature compressed tendon. PRG4 is involved in the congenital joint pathology known as CACP. Analyses of the PRG4 sequence reveal a propensity for a diverse number of functions including lubrication, matrix-binding, self-aggregation, cytoprotection and cell proliferation. This study set out to investigate the potential functions of the N- (exons 2-5) and C-terminal (exons 7-12) structural domains of human and bovine PRG4, facilitated by recombinant protein expression. Preliminary results from solid-phase binding assays showed an interaction between the N- terminal domain of PRG4 and plasminogen activator inhibitor-1 (PAI-1), type II collagen and a 70kDa fibronectin fragment. Immunoprecipitaion experiments demonstrated a potential interaction between the PRG4 C-terminal domain and fibronectin fragments. Both the N- and C- terminal contain functional heparin binding sites. The C-terminal domain was shown to interact with superficial zone chondrocytes, an interaction that was perturbed by heparin. The study also used purified full-length PRG4 from three sources: human recombinant, bovine articular cartilage and bovine tendon. These PRG4 species bound to heparin and displayed similar glycosylation profiles. This study also shows that PRG4 is susceptible to degradation by a number of matrix proteinases including elastase, plasmin, matrix metalloproteinase-7 and cathepsin B. Collectively, the data presented in this thesis provides novel information concerning the biochemistry, susceptibility to digestion and functional capabilities of PRG4.

L'objectif ultime de notre travail etait de developper in vitro des structures cartilagineuses hyalines de taille clinique a partir de cellules competentes et d'un materiau support en acide polyglycolique (pga) cultives dans un bioreacteur rotatoire original (robs). Le robs permet des echanges gazeux au travers d'une fine membrane en silicone. La rotation du systeme ameliore la diffusion des fluides et des nutriments au sein du materiau, et engendre des contraintes hydrostatiques en compression favorables a la production d'une matrice cartilagineuse. Plusieurs parametres etaient evalues : la source cellulaire (chondrocytes, cellules souches mesenchymateuses (csm)), l'age des donneurs (cellules fŒtales, ou adultes), le site donneur (chondrocytes elastiques ou hyalins, csm medullaires ou du sang de cordon ombilical), le materiau support (pga seul, ou associe a un hydrogel). Au terme de ce travail, nous avons pu etablir les conditions de culture de structures cartilagineuses implantables a partir de plusieurs sources cellulaires. Les tissus comportaient un taux de glycosaminoglycanes (gags) identique a celui du cartilage natif, mais une quantite de collagene de type ii significativement inferieure a celle du cartilage hyalin et une quantite notable de collagene de type i. La presence de collagene de type i peut etre expliquee par le caractere tres heterogene de la population cellulaire de depart dont seules 30% peuvent se differencier en chondrocytes. Dans un second temps, les tissus fabriques in vitro a partir de cellules prelevees in utero ont permis de reparer des pertes de substance tracheale, et parietale chez le fŒtus de brebis
The ultimate goal of this work was the fabrication of implantable hyalin-like cartilaginous structures from competent cells seeded onto polyglycolic acid scaffolds (pga) and cultured in a rotating bioreactor system (robs). In the robs, cas exchange was possible through a thin membrane of silicon. The robs supplied optimal oxygen levels and continuous hydrostatic pressure to tissue-engineered structures several parameters were evaluated: cell source (chondrocytes, mesenchymal stem cells (mscs)), donor age (fetal or adult cells), donor site (elastic or hyalin chondrocytes, bone marrow mscs or umbilical coord blood mscs), type of scaffold (pga alone or combined with a hydrogel). Culture conditions were adapted according to cell source. Engineered cartilage constructs exhib1ted similar concentrations of glycosaminoglycans than that of native cartilage, but lower quantitities of type ii collagen and higher levels of type i collagen. The expression of type i collagen may be explained by the heterogenous nature of the bone marrow which contains only 30% of pluripotent cells that can be turned to chondrocytes. Subsequente y, the in vitro engineered constructs were implanted in vivo to repair fetal tracheal and chest wall cartilaginous defects

Knee pain is a common complaint among older Americans, nearly half of whom have developed or will develop painful osteoarthritis. Osteoarthritis is primarily a disease of articular cartilage, the low-friction, shock-absorbing connective tissue that lines long bones at their articulating surfaces. Within these joint tissues and within arthritis, the minor protein collagen VI plays an uncertain role, although it has been implicated in several muscle and ligament disorders. Determination of the collagen VI role in bone and cartilage of the knee is the focus of this dissertation.

Within articular cartilage, collagen VI exclusively localizes to and delimits the pericellular matrix (PCM), which differs from the extracellular matrix (ECM) in composition and structure. To interact with the cell, a molecule must first pass through the PCM. Fluorescent dextran diffusivities were quantified in the cartilage PCM using a newly developed model of scanning microphotolysis (SCAMP), a line photobleaching technique. Diffusion was slower in the PCM than in the ECM, although not in early-stage arthritic tissue. These results support the hypothesis that diffusivity is lower in the PCM than in the ECM of healthy articular cartilage, presumably due to differences in proteoglycan content.

Arthritic degradation is partly mediated by interleukin-1 (IL-1), a catabolic cytokine that affects the mechanical properties of articular cartilage and preferentially binds to cell-surface receptors in the surface zone. Since cells are the cartilage metabolic units, matrix degradation is hypothesized to influence molecular transport in the PCM before the ECM. Cartilage was cultured with or without IL-1, soaked in FITC-ovalbumin, and photobleached using SCAMP to measure diffusivity. Over 7 days of culture, IL-1 doubled the diffusivity in both zones (surface, middle) and matrices (PCM, ECM) of the cartilage. Diffusivity within the PCM was slightly lower than within the ECM. No increase in PCM diffusivity relative to ECM diffusivity was detected within either zone, suggesting that PCM-localized degradation either cannot be distinguished at these time points or cannot be detected by measures of ovalbumin diffusion.

To determine the effects of collagen VI absence on the morphometry and physical properties of the joint, knees of 2-, 9-, and 15-month-old Col6a1+/+ and Col6a1-/- mice were studied. Bone morphometry was evaluated using micro-computed tomography (microCT). Subchondral bone thickness, joint-capsule thickness, and cartilage degradation were assessed by histology. Cartilage elastic modulus, roughness, and coefficient of friction were measured by atomic force microscopy (AFM). Diffusion through the cartilage ECM was determined by SCAMP. Overall, collagen VI absence had profound effects on the morphometry of the proximal tibia and the overall histological structures of the mouse knee, yet minimal effects on the friction, roughness, elastic modulus, and diffusional properties of the articular cartilage. Musculoskeletal abnormalities at the knee do result from collagen VI absence.

Dissertation

Yes
Poly(vinyl alcohol) (PVA)/graphene oxide (GO) nanocomposite hydrogel as artificial cartilage replacement was prepared via freezing/thawing method by introducing polyethylene glycol (PEG). Efficient grafting of PVA molecules onto GO surface was realized by formation of hydrogen bonding, resulting in exfoliation and uniform distribution of GO in PVA matrix. By introduction of appropriate content of GO, the increased crystalline regions of PVA and the formation of GO centered second network structure led to the increase of the storage modulus and effective cross-linking density. And therefore the mechanical strength and toughness of the composite hydrogel were improved simultaneously: the tensile strength, elongation at break, and compressive modulus showed approximately 200%, 40%, and 100% increase of the neat PVA hydrogel. Besides, for the sample with 1.5 wt % GO content, the maximum force retention and dynamic stiffness were improved remarkably in the process of sinusoidal cyclic compression, and the compressive relaxation stress also increased significantly, indicating the enhancement of the compressive recoverable and antifatigue ability, and resistance to compressive relaxation by formation of high load-bearing, dense, and reinforcing double network structure. Moreover, more than 50% decrease in coefficient of friction was obtained for the composite hydrogel, and the worn surface presented relative smooth and flat features with sharp decreasing furrow depth, confirming the lubrication effect of GO-PEG. This study shows promising potentials in developing new materials for cartilage replacement with simultaneous combination of high mechanical property and excellent lubrication.

Dissertação de mestrado, Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014
CRTAC (Cartilage Acidic Protein) firstly identified as a chondrocyte marker in humans and implicated in a number of diseases. This ancient protein is present from prokaryotes to vertebrates and the teleost are the only group that contain duplicates (CRTAC1/CRTAC2). The structure of CRTACs is poorly characterized and was the starting point of the present study. To establish the molecular and structural characterization of CRTAC, three recombinant proteins [human (h) CRTAC1 and sea bass (Dicentrarchus labrax, dl) CRTAC1 and CRTAC2] were over-expressed in E.coli as inclusion bodies and their identity was confirmed by mass spectrometry. The resulting refolded recombinant proteins were obtained with a productivity of 11,51, 8,4 and 11,9 mg of protein per gram of biomass for hCRTAC1, dlCRTAC1 and dlCRTAC2 respectively and approximately 23,48%, 9,09% and 33,46% of, hCRTAC1, dlCRTAC1 and dlCRTAC2 were lost as insoluble aggregates. Size exclusion chromatography revealed the presence of mostly soluble aggregated forms of piscine CRTACs and a mixture of aggregates and monomeric form for hCRTAC1. Spectroscopic studies of human CRTAC1 and sea bass CRTAC1/CRTAC2 showed that all proteins possess secondary and tertiary structure and are particularly rich in β- pleated sheet structure (≈40%), have ≈10,3% alpha-helix and the remainder is disordered. The thermal stability of CRTAC´s structure was assessed considering heating (from 20-90ºC) and freezing (-80ºC). CRTACs retain their native secondary and tertiary structures upon heating, however a slight loss of structure was observed for hCRTAC1 at 60ºC. Freezing induced loss of secondary structure and conformational changes more pronounced for hCRTAC1 and third cycle of dlCRTAC2 and less perceptible for dlCRTAC1. Amyloid formation by CRTACs was assessed in Thioflavin-T assays and a decrease in fluorescence was observed after incubation. In summary, CRTAC´s have propensity to form soluble aggregates that are highly thermostable and these structural properties are conserved from teleosts to humans.
A CRTAC (proteína acídica da cartilagem) é uma proteína da matriz extracelular, inicialmente identificada em humanos como marcador de condrócitos e que está também associada a várias doenças, como a esclerose múltipla. Esta proteína ancestral está presente desde os procariotas até aos vertebrados e os teleósteos são o único grupo que contém o gene duplicado (CRTAC1/CRTAC2). Ambas as proteínas apresentam estrutura similar e possuem na região N-terminal um domínio semelhante ao das cadeias α presente nas integrinas, mas diferem na região C-terminal pelo facto da CRTAC1 ter um domínio adicional descrito como um factor de crescimento epidérmico com ligação a iões Ca2+ . A estrutura conservada das CRTAC indica que estas proteínas podem ter uma função importante que ainda não foi identificada e o facto da sua estrutura estar pouco caracterizada foi o objecto deste estudo. Para o estudo de caracterização molecular e estrutural das CRTAC, três proteínas recombinantes [hCRTAC1 humana e CRTAC1 e 2 de robalo (Dicentrarchus labrax ,dl)] foram sobrexpressas como corpos de inclusão em E.coli e a sua identidade foi confirmada por espectroscopia de massa. Após a solubilização, a purificação e o “refolding”, a produtividade das proteínas recombinantes resultantes foi de, 11,89 para a hCRTAC1, 8,4 para a dlCRTAC1 e 11,9 para a dlCRTAC2, expressa em mg de proteína obtida por g de biomassa. Durante este processo, foram registadas perdas proteicas na forma de agregados insolúveis de aproximadamente 23,48% de hCRTAC1, 9,09% de dlCRTAC1 e 33,46% de dlCRTAC2. Análises de cromatografia de exclusão molecular, das proteínas CRTAC solubilizadas, revelaram a presença de agregados de elevado peso molecular da dlCRTAC1 e 2 de peixes e uma mistura de agregados e monómero da CRTAC1 humana. Os estudos espectroscópicos, demonstraram que, as proteínas recombinantes CRTAC têm estrutura secundária e terciária e que a sua estrutura secundária é particularmente rica em folha-β (≈40%), tendo apenas 10,3% de hélice-α e a restante estrutura é desordenada. Neste estudo foi avaliada a estabilidade térmica da estrutura das CRTAC, considerando o efeito do aquecimento (de 20º a 90ºC) e do congelamento em vários ciclos (-80ºC). Os resultados indicaram que, as CRTAC mantêm a sua estrutura secundária e terciária após o aquecimento, no entanto, no caso da CRTAC1 humana foi observada uma ligeira perda de estrutura a partir dos 60ºC. O efeito do congelamento induziu a uma perda de estrutura secundária e também a alterações conformacionais mais evidentes na CRTAC1 humana relativamente á dlCRTAC1 e dlCRTAC2 de peixes. A capacidade de formação de fibrilas amilóides pelas CRTAC, sob certas condições de incubação, foi avaliada utilizando ensaios com Tioflavina-T. Após a incubação foi observada uma diminuição da fluorescência e estes resultados sugerem que, as CRTAC´s podem facilmente formar agregados de grandes dimensões com propriedades não-amilóides que, podem ser degradados ou sair de solução após longos periodos de incubação. Admite-se a hipótese de que tais agregados possam ser estruturas funcionais. Resumindo, as proteínas CRTAC tendem a formar agregados solúveis que são altamente termoestáveis e estas propriedades estruturais são conservadas desde os teleósteos até aos humanos

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Articular cartilage functions successfully as a compression load-bearing tissue by virtue of the functional interplay between a 3-dimensional structure of collagen fibrils and the entrapped water-swollen proteoglycan molecules. Crucial to this entrapment process is a mechanism or set of mechanisms that maintain the collagen fibrils in a finely divided interconnected configuration that immobilises the macro-molecular proteoglycan complexes. Any loss of interconnectivity in the collagen network that might reduce the constraints on the swelling tendency of the proteoglycan domains will lead to a lower matrix stiffness. There are some structural similarities between this less stiff or abnormally softened cartilage and the degenerative osteoarthritic matrix, although ultrastructural studies to date are somewhat limited. The primary objective of the research reported in this thesis was to investigate the fibrillar architecture in the general matrix of both the normal and abnormally softened cartilage matrices. The fibrillar architectures of the normal and abnormally softened general matrices were compared using Nomarski light microscopy (LM), transmission electron microscopy (TEM) with combined stereoscopic reconstruction, and scanning electron microscopy (SEM). As reported earlier by Broom (1984b), a pseudo-random network developed from an overall radial arrangement of collagen fibrils is the most fundamental ultrastructural characteristic of the normal general matrix. By contrast, this present investigation has shown that the most distinctive feature of the softened matrix is the presence of parallel and relatively unentwined fibrils, strongly aligned in the radial direction. A structural model illustrating the transformation from the normal to the softened matrix is proposed based on the important property of lateral interconnectivity in the fibrils which involves both entwinement and non-entwinement based interactions. The distribution of proteoglycans in the normal and the softened matrix was compared. The distribution of Type II collagen was investigated using immunohistochemical staining combined with confocal imaging. It is concluded that the Type II fibrils do persist in the altered matrix thus adding further experimental support for the proposed transformation model. The swelling behaviour of the general matrix of both normal and abnormally softened articular cartilage was compared by subjecting tissue specimens under different modes of constraint to a high swelling bathing solution of distilled water and comparing structural changes imaged at the macroscopic, microscopic and ultramicroscopic levels of resolution. Near-zero swelling was observed in the isolated normal general matrix with minimal structural change. By contrast, the similarly isolated softened general matrix exhibited large-scale swelling in both the transverse and radial directions. This difference in dimensional stability was attributed to fundamentally different levels of fibril interconnectivity between the two matrices. The structural transformation model was further developed to accommodate fibrillar rearrangements associated with the large-scale swelling in the radial and transverse directions in the softened general matrix.