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.

Made available in DSpace on 2017-07-10T17:48:09Z (GMT). No. of bitstreams: 1 Douglas-Fernando-Bayerle.pdf: 1438510 bytes, checksum: 2daffbb020be65c4b31cd27c080ea2a4 (MD5) Previous issue date: 2015-04-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The present work aimed to evaluate the use of tannins as adsorbent of heavy metals in broiler chicks fed diets with different levels of mussel flour inclusion in replacement of limestone of the feeding. Two experiments were divided in two phases with two assays each. In the first experiment we conducted a trial to evaluate the performance of broiler chicks from 1 to 21 days fed wattle tannin, using for it 720 Cobb-500 1-day-old male broiler chicks distributed in a completely randomized design with six treatments and six replicates of 20 birds in each experimental unit. The treatments were composed by basal diets with increasing levels of wattle tannin (0, 250, 500, 750, 1000, 1250 g/ton-1 of wattle tannin). The results showed that the use of 250 g/ton-1 of tannin was detrimental to performance, and morphometric measurements suffered no statistical difference using tannin in the diet. In the second trial, we evaluated the performance of broiler chicks from 1 to 21 days of age fed increasing levels (0, 25, 50, 75, 100%) of replacement of mussel meal by limestone in feeding and still supplementation or not of wattle tannin (250 g/ton-1 of wattle tannin) with 5 replicates per treatment. For that, 1200 Cobb-500 1-day-old male broiler chicks were housed in a completely randomized design, with 24 birds each. The results showed that the golden mussel has lot of calcium, low levels of heavy metals, and microbiological contamination did not present or is below the amounts allowed by law. For performance, the data demonstrated that the use of mussel can be performed up to 100% replacement of the limestone in feeding without affecting the variables, but the use of 250 g/ton-1 of wattle tannin was detrimental to the performance of broiler chicks. There was no influence on the use of mussel and tannin on the deposition of heavy metals in the body of birds. The variables, liver and kidneys showed the interaction between the tested diets. The bony features, just gray on fresh matter showed interaction between treatments. In the second experiment, the first test was to evaluate the performance of broiler chickens to the stage 21-42 days, fed with wattle tannin. For that, 648 Cobb-500 21-days-old male broilers were distributed in a completely randomized design, divided into six treatments and six replicates of 18 birds in each experimental unit. The treatments were composed by basal diets with increasing levels of wattle tannin (0, 250, 500, 750, 1000, 1250 g/ton-1 of wattle tannin). The data showed that the use of levels above 500 g / t of wattle tannin in the diet is detrimental to the performance of the birds, and there were no statistical differences in morphometric measures. Carcass yield decreased linearly with the addition of tannin in the diet and there was a decrease in fat deposition, and an increase in liver size. In the second trial, the performance of broilers from 21 to 42 days was evaluated. For that, 900 Cobb-500 21-days-old male broilers were housed in a completely randomized design, with increasing levels of substitution (0, 25, 50, 75, 100%) of the golden mussel flour in replacement of limestone and also with supplement or not of wattle tannin (250 g/ton-1 of wattle tannin) with 5 replicates per treatment and 18 birds in each experimental unit. The performance showed that the use of mussel can be performed up to 100% in replacement of the limestone in the feeding without affecting the variables, but the use of 250 g/ton-1 of wattle tannin was detrimental to the performance of broiler chickens cutting. For bony features, the use of tannin decreased bone strength (kgf) and increased phosphorus in natural matter, and there was an interaction between variables to Seedor index and gray on fresh matter. The use of tannin negatively affected carcass yield and the relative weight of kidneys, and there was a significant interaction for yield wings and legs. Calcium levels in the blood suffered interaction between the substitution levels studied / O desenvolvimento deste trabalho teve como objetivo avaliar a utilização de taninos como adsorvente de metais tóxicos em rações de pintos de corte alimentados com diferentes níveis de inclusão de farinha de mexilhão dourado em substituição ao calcário calcítico das rações. Foram realizados dois experimentos divididos em duas fases com dois ensaios cada, no primeiro experimento foi realizado um ensaio para avaliar o desempenho de pintos de corte para fase 1 a 21 dias, alimentados com tanino de acácia, utilizando-se 720 pintos de corte, machos, com um dia de idade, Cobb 500, distribuídos em um delineamento inteiramente casualizado, com seis tratamentos e seis repetições, com 20 aves em cada unidade experimental. Os tratamentos eram formados por rações basais com níveis crescentes de tanino de acácia (0, 250, 500, 750, 1000, 1250 g/ton-1 de tanino de acácia). Os resultados demostraram que a utilização de 250 g/ton-1 de tanino foi prejudicial para o desempenho, e as medidas morfométricas não sofreram diferença estatística utilizando tanino na dieta. Num segundo ensaio foi avaliado o desempenho de pintos de corte para a fase 1 a 21 dias de idade alimentados com níveis crescente (0; 25; 50; 75; 100%) de substituição de farinha de mexilhão dourado pelo calcário calcítico da ração e ainda a suplementação ou não de tanino de acácia (250 g/ton-1 de tanino de acácia), com 5 repetições por tratamento. Foram alojados em delineamento inteiramente casualizado, 1200 pintos de corte, machos, com um dia de idade, Cobb 500, com 24 aves por unidade experimental. Os resultados apontaram que o mexilhão dourado possui grande quantidade de cálcio, níveis baixíssimos de metais tóxicos, e a contaminação microbiológica não apresentou ou encontra-se abaixo dos valores permitidos pela legislação vigente. Para o desempenho, os dados demonstraram que a utilização do mexilhão dourado pode ser realizada em até 100% de substituição ao calcário calcítico das rações sem afetar as variáveis estudadas, porém a utilização de 250 g/ton-1 de tanino de acácia foi prejudicial para o desempenho dos pintos de corte. Não foi observado influencia na utilização de mexilhão dourado e tanino sobre a deposição de metais tóxicos no organismo das aves. As variáveis, fígado e rins, apresentaram interação entre as dietas avaliadas. As características osseas, apenas a cinza na matéria natural apresentou interação entre os fatores estudados. No segundo experimento, o primeiro ensaio teve como objetivo avaliar o desempenho de frangos de corte para a fase de 21 a 42 dias, alimentados com tanino de acácia. Aos 21 dias foram distribuídos em um delineamento inteiramente casualizado, com 648 frangos de corte, machos, Cobb 500 divididos em seis tratamentos e seis repetições, com 18 aves em cada unidade experimental. Os tratamentos eram formados por rações basais com níveis crescentes de tanino de acácia (0, 250, 500, 750, 1000, 1250 g/ton-1 de tanino de acácia). Os dados encontrados demostraram que a utilização com níveis acima de 500 g/ton-1 de tanino de acácia na dieta é prejudicial para o desempenho das aves, e não foram encontradas diferenças estatísticas para as medidas morfométricas. O rendimento de carcaça decresceu linearmente com a inclusão de tanino na dieta, e houve uma diminuição na deposição de gordura, e um aumento no tamanho do fígado. No segundo ensaio foi avaliado o desempenho de frangos de corte para a fase de 21 a 42 dias, alojados em um delineamento inteiramente casualizado, com níveis crescente de substituição (0; 25; 50; 75; 100%) de farinha de mexilhão dourado pelo calcário calcítico da ração e ainda a suplementação ou não de tanino de acácia (250 g/ton-1 de tanino de acácia), com 5 repetições por tratamento. Aos 21 dias as aves foram distribuídas em um delineamento inteiramente casualizado, 900 frangos de corte, machos, Cobb 500, com 18 aves em cada unidade experimental. O desempenho nesta fase demonstrou que a utilização do mexilhão dourado pode ser realizada em até 100% de substituição ao calcário calcítico das rações sem afetar as variáveis estudadas, porém a utilização de 250 g/ton-1 de tanino de acácia foi prejudicial para o desempenho dos frangos de corte. Para as características osseas, a utilização de tanino diminuiu a resistência óssea (kgf) e aumentou o fósforo na matéria natural, e houve interação entre as variáveis estudadas para o índice Seedor e cinza na matéria natural. A utilização de tanino afetou negativamente o rendimento de carcaça e o peso relativo dos rins, e houve interação significativa para o rendimento de asas e pernas. Os níveis de cálcio no sangue sofreram interação entre os níveis de substituição estudados

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Mechanical loading associated with exercise is known to benefit bone health; however, most studies explore exercise benefits on bone mass independent of bone structure and strength. The purpose of this dissertation is to explore the response of the skeleton to exercise across the translational divide between animal- and human-based studies, with a particular emphasis on exercise-induced changes in bone structure and estimated strength. To explore the skeletal benefits of exercise, models were used wherein loading is introduced unilaterally to one extremity. Unilateral exercise enables the contralateral, non-exercised extremity to be used as an internal control site for the influences of systemic factors, such as genetics and circulating hormones. In study 1, a dose response between load magnitude and tibial midshaft cortical bone adaptation was observed in mice that had their right tibia loaded in axial compression at one of three load magnitudes for 3 d/wk over 4 weeks. In study 2, the ability of peripheral quantitative computed tomography to provide very good prediction of midshaft humerus mechanical properties with good short-term precision in human subjects was demonstrated. In study 3, collegiate-level jumping (long and/or high jump) athletes were shown to have larger side-to-side differences in tibial midshaft structure and estimated strength between their jump and lead legs than observed in non-jumping athletes. In study 4, prepubertal baseball players followed for 12 months were shown to gain more bone mass, structure and estimated strength in their throwing arm relative to their nonthrowing arm over the course of 12 months. These cumulative data using a combination of experimental models ranging from animal to cross-sectional and longitudinal human models demonstrate the ability of the skeleton to adapt its structure and estimated strength to the mechanical loading associated with exercise. Study of these models in future work may aid in optimizing skeletal responses to exercise.

Es erfolgt eine Knochenstrukturanalyse der proximalen Tibia unter Berücksichtigung verschiedenster radiologischer, biomechanischer und histomorphometrischer Aspekte. Die regionen-, alters- und geschlechtsspezifischen Aspekte dieser Problemregion werden herausgearbeitet. Der eindeutige Nachweis einer regionen-abhängigen Verteilung der Knochendichte und der biomechanischen Eigenschaften in der proximalen Tibia ist eines der Hauptergebnisse der vorliegenden Studie. In der proximalen Tibia besteht eine signifikante Abnahme der Knochendichte von proximal nach distal. Im zentralen Bereich der proximalen Tibia besteht in allen Sektionen im Vergleich zu den anterior/posterior und medial/lateral liegenden Gebieten die niedrigste Knochendichte. In der vorliegenden Studie wurde die proximale Tibia in 3 Etagen (von proximal nach distal) unterteilt. Beim Vergleich der auf diesen Etagen aufgebrachten ROIs (region of interest,jeweils 5 in den beiden proximalen Etagen und 4 im distalen Abschnitt) zeigte sich in den beiden proximalen Etagen lateral (Ebene I anterolateral/ Ebene II posterolateral) die höchste Knochendichte. Im Gegensatz dazu zeigte sich in der distalen Etage anteromedial die höchste Knochendichte. Weiterhin wurden die 3 gängigen Stabilisierungsverfahren für diese Region einer umfangreichen biomechanischen Testung unterzogen. Es zeigte sich, dass der Ilizarov Fixateur bei den verschiedensten Lastfällen meist das instabilste Implantat war. Trotz der biomechanischen Defizite konnten die in der klinischen Studie mit Composite Fixateur versorgten Frakturen trotz erheblichem Weichteilschaden und instabiler Fraktursituation zur Ausheilung gebracht werden. Das LIS-System erwies sich gegenüber der konventionellen Abstützplatte hinsichtlich der biomechanischen Steifigkeit sowohl in der statischen als auch in der zyklischen Testung als gleichwertiges oder sogar biomechanisch günstigeres Implantat. Diese positiven klinischen wie biomechanischen Erfahrungen führen auch zur Förderung der Entwicklung anderer winkelstabiler Fixateur interne-Systeme in den verschiedensten Problemregionen (Pilon tibiale, proximaler und distaler Humerus, distaler Radius). Als wesentliche neue Therapieansätze für das operative Vorgehen in der Problemregion der proximalen Tibia lassen sich die folgenden Gesichtspunkte herausarbeiten: 1) Knochendichteadaptierte Implantat- und Schraubenpositionierung bei der konventionellen Osteosynthese, 2) Knochendichteadaptierte Pin- und Olivendrahtpositionierung bei externen Fixationsverfahren (Ilizarovringfixateur, Fixateur externe) im Bereich der proximalen Tibia, 3) Implantatverbesserungen (LISS-Schraubenkonfiguration und -positionierung, Plattendesign, Umstellungsplatte, Verriegelungsbolzen bei Marknägeln wie UTN, PTN), 4) Prothesenverbesserung (knochendichteadaptiertes Zapfendesign mit 3 Zapfen für die tibiale Komponente). / In this study an analysis of the bone structure of the proximal tibia was performed with special attention paid to the different radiological, biomechanical and histomorphometrical aspects. Region-, age- and gender-specific attributes of the localised bone were also examined. Evidence of a region related variation of bone density and biomechanical behaviour is one of the main results of this study. In the proximal tibia, a significant reduction in the bone density exists from proximal to distal. In comparison to the anterior/posterior or medial/lateral areas, the lowest bone mineral densities were found in the central region. In this study the proximal tibia was divided into 3 different levels (from proximal to distal). When comparing the different regions of interest (ROIs) 5 each in the two proximal levels and 4 in the most distal level), the lateral regions (level 1 anterolateral/ level 2 posterolateral) presented the highest bone mineral density. In contrast, the highest bone density in the distal- level was detected in the anteromedial region. Furthermore, complex biomechanical testing of- 3 common fixation techniques for fracture situations of the proximal tibia was performed. It was shown that the Ilizarov fixator was the most unstable implant in several load tests. Despite this biomechanical deficit fractures treated by composite- fixators in different clinical trails healed uneventfully, even with severe soft tissue damage or an unstable fracture situation. In comparison to the conventional buttress plate, the LIS-System was an equal or superior implant, both in static and cyclic stiffness testing. These clinical and biomechanical experiences lead to the development of other angle stable internal fixator systems for different problematic regions (tibial plafond, proximal and distal humerus, distal radius). The following new therapeutic aspects were developed for the surgical treatment of the proximal tibia: 1) Bone mineral density adapted implant-and screw placement in conventional plating. 2) Bone density adapted pin- and olive wire placement during external fixation (ilizarov ring fixator, external fixator) techniques of the proximal tibia. 3) Improvement of implant design (LISS screw configuration and- placement, plate design, locking bolt configuration in nails). 4) Improvement of prosthetic design (bone density adapted design of the tibial components)