Bioreactors are devices used for the growth of tissues in a laboratory environment. They exist in many different forms, each designed to enable the production of high-quality tissues. The dynamic environment within bioreactors is known to significantly affect the growth and development of the tissue. Chondrocytes, the building blocks of articular cartilage, for example, are stimulated by mechanical stresses such as shear, as compared with those in tissues grown under static incubation conditions. On the other hand, high shear can damage cells. Consequently the shear-stress level has to be controlled in order to optimize the design and the operating conditions of bioreactors. Spinner flasks have been used for the production of articular cartilage in vitro. Assuming the existence of a relation between the cellular glycosaminoglycan (GAG) synthesis and the local shear stresses on the construct surfaces, this research focuses on the development of a model for cartilage growth in such devices. The flow produced in a model spinner flask is characterized experimentally using particle-image velocimetry (PIV). A computational fluid dynamic (CFD) model validated with respect to the laboratory measurements is constructed in order to predict the local shear stresses on the construct surfaces. Tissue growth experiments conducted in the prototype bioreactor permit construct histologies and GAG contents to be analyzed and then correlated with the shear-stress predictions. The integration of this relation into the CFD model enables the prediction of GAG synthesis through convective effects. Coupling this convective model to an existing diffusive model produces a complete cartilage-growth model for use in aiding the optimization of existing bioreactors, and in the design of new ones.

Solute diffusion is critical to maintenance of cellular function and matrix integrity in articular cartilage. Nutrient deficiency due to transport limitations is thought to be one of the causes of the pathological degeneration of the cartilage tissue. Thus, a study of diffusion within cartilage will lead to a better understanding of the causes of cartilage degeneration. To accurately estimate diffusion coefficients in cartilage and other hydrated medium, we developed a finite-element based method, the Direct Diffusion Simulation Parameter Estimation method (DDSPE), to be used for quantitative determination of solute diffusivities from Fluorescence Recovery After Photobleaching data. Analyses of simulated and experimental FRAP data demonstrated that this method was more accurate than existing analytical methods, including having a low sensitivity to variations in the spot radius. Subsequently, the roles of extracellular matrix (ECM) composition and tissue orientation in solute diffusion within immature bovine cartilage were explored. Diffusivities were measured through the cartilage depth and in two different orientations (radial and transverse). Diffusivities were then correlated with ECM components. Matrix water content was found to be the best predictor of solute diffusion rates in immature cartilage. Although no specific experiments were done to measure the effect of structure, our results suggested that matrix structure did indeed modulate transport. Diffusional anisotropy, defined as the ratio of the diffusivities in both orientations, was observed to be significant in all the immature cartilage zones. As a consequence, the differences in solute diffusion between immature and mature bovine cartilage were investigated. Diffusion rates and diffusional anisotropy decreased in the mature cartilage superficial zone. The decrease in diffusivities observed in mature cartilage suggests that there may be a reduction in nutrient and growth factor supply to the cells. Nevertheless, healthy adult cartilage can still maintain its normal function even with a reduction in solute diffusion rates as nutrient diffusion distances are shorter in mature cartilage. However, any disruption in the mechanical or biological environment could cause an imbalance in tissue homeostasis, which when combined with decreased diffusivities, could trigger matrix degeneration. Thus, decreased diffusivity may be a necessary but not a sufficient prerequisite of matrix degeneration.

L’objectif de cette étude est de vérifier si un ciment ostéoconducteur résorbable utilisé comme technique de fixation de greffons ostéochondraux permet d'obtenir une stabilité initiale supérieure à celle obtenue avec la technique de mosaicplastie originalement décrite. Il s’agit d’une étude biomécanique effectuée sur des paires de fémurs cadavériques bovins. Pour chaque paire de fémurs, des greffons ostéochondraux autologues ont été insérés et stabilisés au moyen d’un ciment biorésorbable (Kryptonite, DRG inc.) sur un fémur alors qu’au fémur controlatéral, les greffons ont été implantés par impaction selon la technique usuelle de mosaicplastie. Des greffons uniques ainsi que des greffons en configuration groupée ont été implantés et soumis à une évaluation biomécanique. Les charges axiales nécessaires pour enfoncer les greffons de 1, 2 et 3 mm ont été comparées en fonction de la technique de stabilisation utilisée, ciment ou impaction, pour chaque configuration de greffons. Les résultats démontrent que les greffons ostéochondraux cimentés uniques et groupés ont une stabilité initiale supérieure à celle de greffons non cimentés sur des spécimens cadavériques bovins. L’obtention d’une plus grande stabilité initiale par cimentation des greffons ostéochondraux pourrait permettre une mise en charge précoce post-mosaicplastie et mener à une réhabilitation plus rapide. / The objective of this project is to compare the primary stability of osteochondral autografts stabilized with a resorbable osteoconductive bone cement to that of bottomed press fit grafts inserted according to the original mosaicplasty technique. Biomechanical testing was conducted on pairs of cadaveric bovine femurs. For each femoral pair, osteochondral grafts were inserted and stabilized with an osteoconductive bone cement (Kryptonite, DRG inc.) on one bone whereas on the controlateral femur, grafts were inserted in a press fit fashion. Grafts were inserted in 2 different configurations, single grafts as well as groups of 3 adjacent grafts, and submitted to biomechanical testing. Axial loads needed to sink the grafts to 1, 2 and 3 millimeters below cartilage level were recorded and compared according to the fixation technique, cement or press-fit impaction, for each graft configuration. According to those results, cemented osteochondral autografts appear more stable than press fit grafts for both single and 3-in-a-row configurations. Using such a cementation technique could potentially prevent the initial loss of stability that has been shown to occur with osteochondral grafts in the post-operative period, allowing patients to perform early weight bearing and rehabilitation.

Hyaline cartilage is a highly specialized tissue, which plays an important role in the articulating joints of an individual. It provides the joints with a nearly frictionless, impact resisting surface to protect the ends of the articulating bones. Articular cartilage has a poor self-repair capacity and, therefore, it rarely heals back to normal after an injury. Overweight, injuries, overloading and genetic factors may initiate a degenerative disease of the joint called osteoarthritis. Osteoarthiritis is a major global public health issue. Currently, the most used treatment for large articular cartilage defects is joint replacement surgery. However, possibilities to replace this highly invasive operation with strategies based on tissue engineering are currently investigated. The idea of the tissue engineering is to optimize the use of the cells, biomaterials and culture conditions to regenerate a new functional tissue for the defect site. The goal of this thesis was to manufacture cartilage tissue in cell culture conditions in vitro. Bovine primary chondrocytes isolated from the femoral condyles were used in all the experiments for neocartilage production. The samples were collected for histology, gene expression level quantifications, and analyses of proteoglycan (PG) content and quality. The histological sections were stained for type II collagen and PGs, the quantitative RT-PCR was used to observe the relative expressions of aggrecan, Sox9, procollagen α2(I) and procollagen α1(II) genes. The PGs were quantified using a spectrophotometric method, and agarose gel electrophoresis was used to separate the PGs according to their size. In the two first studies, we optimized the culture conditions of in vitro scaffold-free culture technique to produce the native-type hyaline cartilage of a good quality. We found out that high glucose concentration and hypertonic medium at 20% oxygen tension promoted the best hyaline-like neocartilage tissue production. Glucosamine sulfate supplementation, low oxygen tension, 5 mM glucose concentration and a transient TGF-β3 supplementation were not beneficial for the neocartilage formation in the scaffold-free cell culture system. In the third study, we used these newly defined, optimized culture conditions to produce the neocartilage tissues in the HyStem™ and the HydroMatrix™ scaffold materials and we compared these tissues to the ones grown as scaffold-free control cultures. We noticed that there was no difference between the controls and the scaffolds, and occasionally the scaffold-free controls had produced better quality cartilage than the ones with the scaffolds. Overall, the neocartilage tissues were of good hyaline-like quality in the third study. Their extracellular matrix contents were close to the native cartilage, although the neotissues lacked the zonal organization typical to the normal articular cartilage. The tissues had the right components, but their ultrastructure differed from the native cartilage. In conclusion, we were able to optimize our in vitro neocartilage culture method further, and discovered a good combination of the culture conditions to produce hyaline-like cartilage of good quality. Surprisingly, the scaffold materials were not beneficial for the cartilage formation. / Lasi- eli hyaliinirusto on pitkälle erikoistunutta kudosta, jolla on erittäin tärkeä rooli yksilön nivelten toiminnassa. Kudos suojaa ruston alapuolista luuta muodostamalla lähes kitkattoman ja joustavan liikkumista helpottavan pinnan. Lasiruston oma uusiutumiskyky on hyvin heikko, ja näin ollen kudos vain harvoin paranee alkuperäisen kaltaiseksi vaurion jälkeen. Ylipaino, vammat, liiallinen kuormitus tai geneettiset tekijät voivat käynnistää rustokudoksen rappeutumisen. Tätä tilaa kutsutaan nivelrikoksi. Nivelrikko on valtava kansanterveydellinen ongelma. Keinonivelleikkaus on nykyisellään ainoa hoitokeino pinta-alaltaan laajojen nivelruston vaurioiden hoitoon. Vaihtoehtoja tämän suuren ja invasiivisen kirurgisen operaation korvaamiseksi tutkitaan kuitenkin koko ajan ympäri maailmaa. Kudosteknologian ajatuksena on optimoida solujen, biomateriaalien ja erilaisten kasvatusolosuhteiden käyttö uuden, alkuperäisen kaltaisen toiminnallisen kudoksen luomiseksi vauriokohtaan. Väitöskirjan kaikissa kolmessa osatutkimuksessa uudisrustokudoksia tuotettiin käyttäen naudan polven rustosta eristettyjä primäärisiä rustosoluja. Näytteet kerättiin histologisia analyysejä, geenin ilmentymistutkimuksia ja proteoglykaanisisällön ja -jakauman (PG) analyyseja varten. Histologisista leikkeistä värjättiin tyypin II kollageeni ja PG:t, ja kvantitatiivista RT-PCR -menetelmää käytettiin aggrekaani-, Sox9-, prokollageeni α2(I)- ja prokollageeni α1(II)-geenien suhteellisten ilmentymistasojen määrittämiseen. Proteoglykaanisisältö analysoitiin käyttäen spektrofotometristä menetelmää, ja PG:t eroteltiin kokonsa perusteella agaroosigeelielektroforeesia käyttäen. Kahdessa ensimmäisessä osatutkimuksessa optimoitiin tukirakenteetta kasvattujen uudisrustojen kasvatusolosuhteita natiivin kaltaisen lasiruston tuottamiseksi. Havaitsimme, että korkea glukoosipitoisuus ja hypertoninen elatusaine yhdistettynä 20 % happiosapaineeseen tuotti parhaimman laatuista uudisrustokudosta tutkituista yhdistelmistä. Glukosamiinisulfaatin lisäys, matala happiosapaine, 5 mM glukoosi konsentraatio tai TGF-β3:n lisääminen alkuvaiheessa eivät edesauttaneet uudisrustokudosten muodostumisessa. Kolmannessa osatutkimuksessa otettiin käyttöön uudet, hyväksi havaitut kasvatusolosuhteet yhdistettynä HyStem™ and HydroMatrix™ -tukimateriaaleihin, ja niitä verrattiin tukirakenteettomaan kasvatusmenetelmään. Tutkimuksessa havaittiin, ettei tukirakenteettoman kontrollin tai tukimateriaalien välillä ollut mitään eroa, ja että kontrollikasvatukset tuottivat ajoittain jopa parempaa rustoa kuin tukimateriaalein kasvatetut. Kaiken kaikkiaan kaikki tuotetut uudiskudokset muistuttivat laadullisesti lasiruston kaltaista kudosta. Molekyylisisältö lähenteli natiivia rustoa, vaikkakin uudiskudoksista puuttui normaalille nivelrustolle tyypillinen vyöhykkeinen järjestäytyminen. Kudoksissa oli parhaimmillaan oikea määrä oikeita komponentteja, mutta ne eivät vain olleet järjestäytyneet oikealla tavalla. Onnistuimme optimoimaan uudisrustokudosten kasvatusmenetelmäämme. Löysimme hyvän kasvatusolosuhteiden yhdistelmän, jonka avulla kykenimme tuottamaan lasiruston kaltaista uudisrustokudosta. Hivenen yllättäenkin, tukimateriaalit eivät olleet avuksi tutkimuksessamme uudisrustokudoksia muodostettaessa.

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 2016-06-02T20:18:12Z (GMT). No. of bitstreams: 1 3010.pdf: 1781268 bytes, checksum: 812b3af521e82fd881dcbddfcb1454b8 (MD5) Previous issue date: 2010-03-29 / Universidade Federal de Sao Carlos / The function of articular cartilage depends on the chondrocytes and on the components of the extracellular matrix, which in turn may be regulated by mechanical stimuli. Thus, changes in load support may affect its composition or its structure and interfere with their functional ability to sustain and distribute loads and minimize the stresses of contact. Thus, investigations of articular cartilage components, such as chondrocyte and or matrix components are essential for prevention and treatment of arthritic disease. A greater understanding of the relationship of use / disuse and degeneration as well as the consequences of situations such as shear stress, static load or unloading can generate in this tissue. The aim of this study was to evaluate the response of chondrocytes, proteoglycan, collagen and fibronectin in articular cartilage after application of a protocol of immobilization, stretching and joint remobilization. Material and Methods: We used 36 animals divided into six groups (n = 6): immobilized (I), immobilized and stretched seven days per week (IS7), immobilized and stretched three days per week (IS3), stretched seven days per week (S7), stretched three days per week (S3) and control (C). Groups I, IS3 and AS7 underwent four weeks of immobilization of the left hind limbs. Groups IS7 and IS3, after immobilization, were subjected to three weeks of the posterior muscle stretching of the left hind leg daily or three times per week, respectively. The S3 and S7 groups remained free in the cage for 4 weeks and subsequently underwent three weeks of posterior muscle stretching of the left hind limb daily or three times per week, respectively. After these procedures, the left ankle were collected, decalcified, processed in paraffin and stained with H&E, Safranin-O, Picrossiruius Red and immunostained with fibronectin and chondroitin sulfate 4 for further analysis. Two observers evaluated parameters such as chondrocyte cloning, loss of proteoglycan content, thin and thick fibrils collagen content, intensity of staining for fibronectin and chondroitin sulfate 4. For statistical analysis we used the following tests: Kruskal Wallis and post hoc Newman Keuls: cloning and the proteoglycan content of the different groups); Duncan multiple comparison: morphometric evaluation of cellularity; ANOVA and post hoc Tukey: proportion of thin and thick fibrils of collagen. For analysis of the immunohistochemistry reactions of fibronectin and chondroitin sulfate 4 it was used nonparametric test Kruscal Wallis and post hoc Newman Keuls. In all tests the significance level was p ≤ 0.05. Results: With respect to the cellularity IS7 group showed significant increase in cellularity compared to groups I and C. The IS3 group also showed significant celullar change with the formation of chondrocyte cloning compared to groups S7, S3 and C. The most significant loss of proteoglycan was in IS7 group compared to all other groups. The I group also lost significantly more proteoglycan than the others, except for IS7 group. With respect to collagen fibrils was observed that immobilization (I) significantly reduced the thin fibrils in relation to groups IS3, S7, S3 and C. The quantity of thick fibrils was influenced by mechanical overload, as there was a significant decrease of it in all groups compared to control. With respect to the findings of the fibronectin, the groups immobilized and stretched (IS3 and IS7) had significantly higher intensity staining of fibronectin than other groups. There was no statistical difference of chondroitin sulfate 4 immunostaining among the different groups. Conclusion: The protocols of muscle stretching after immobilization, applied on alternate days and daily provoked distinct adaptive responses in articular cartilage. The immobilization stimulated tissue atrophy that when stimulated by muscle stretching on alternate days, kept some matrix components, such as fine fibrils of collagen and proteoglycan, unlike the protocol used daily. Thus we can conclude that muscle stretching applied in previously immobilized joints should be applied with caution, on alternate days of mechanical stimulation. / A função da cartilagem articular é dependente do condrócito e dos componentes de sua matriz extracelular, que por sua vez, podem ser regulados por estímulos mecânicos. Assim, alterações no suporte de carga podem afetar sua composição ou sua estrutura e interferir na sua capacidade funcional de sustentar e distribuir cargas e minimizar os estresses de contato. Desta forma, investigações dos componentes da cartilagem articular, como o condrócito e ou componentes da matriz são essenciais para prevenção e tratamento de doenças articulares. É necessário um maior entendimento das relações de uso/desuso e degeneração, assim como das conseqüências que situações como estresse de cisalhamento, carga estática prolongada ou ausência de carga possam gerar neste tecido. O objetivo do presente estudo foi avaliar a resposta do condrócito, proteoglicana, colágeno e fibronectina da cartilagem articular, após aplicação de um protocolo de imobilização, alongamento e remobilização articular. Material e Métodos: foram utilizados 36 animais divididos em 6 grupos (n=6): imobilizado (I), imobilizado e alongado 7 dias por semana (IA7), imobilizado e alongado 3 dias por semana (IA3), alongado 7 dias por semana (A7), alongado 3 dias por semana (A3), e controle (C). Os grupos I, IA7 e IA3 foram submetidos a 4 semanas de imobilização da pata traseira esquerda. Os grupos IA7 e IA3, após a imobilização, foram submetidos a 3 semanas de alongamento da musculatura posterior da pata traseira esquerda diariamente ou 3 vezes por semana, respectivamente. Os grupos A7 e A3 permaneceram livres na gaiola por 4 semanas e posteriormente foram submetidos a 3 semanas de alongamento da musculatura posterior da pata traseira esquerda diariamente ou em dias alternados, respectivamente. Após esses procedimentos, os tornozelos esquerdos foram coletados, descalcificados, processados em parafina e corados com H&E, Safranina, Picrossiruius Red e imunomarcados para fibronectina e sulfato de condroitina 4 para posterior análise. Foram avaliados por dois observadores parâmetros como: celularidade, contagem de clones, perda de proteoglicanos, conteúdo de fibrilas finas e grossas de colágeno e expressão de fibronectina e sulfato de condroitina 4. Para comparação destes parâmetros entre os diferentes grupos foram utilizados os seguintes testes estatísticos: Kruskal Wallis com post hoc Newman Keuls: formação de clones e conteúdo de proteoglicanas; Comparações múltiplas de Duncan: avaliação morfométrica de celularidade e Anova com post hoc de Tukey: proporção das fibrilas finas e grossas de colágeno. Para análise das reações de imunohistoquímica para fibronectina e sulfato de condroitina 4 foi utilizado o teste não paramétrico de Kruscal Wallis e post hoc Newman Keuls. Em todos os testes o nível de significância foi de p≤0,05. Resultados: com relação a celularidade o grupo IA7 apresentou aumento significativo da celularidade em relação aos grupos I e C. O grupo IA3 também apresentou alteração celular significativa com formação de clones em relação aos grupos A7, A3 e C. A maior perda significativa de proteoglicanas foi do grupo IA7 em relação a todos os outros grupos. O grupo I também perdeu significativamente mais proteoglicanas que os demais, somente não com relação ao grupo IA7. Com relação às fibrilas colágenas foi observado que a imobilização (I) reduziu significativamente as fibrilas finas em relação aos grupos IA3, A7, A3 e C. Já a quantidade de fibrilas grossas sofreu influência da sobrecarga mecânica, pois que houve diminuição significativa das mesmas em todos os grupos em relação ao controle. Com relação aos achados de fibronectina, os grupos imobilizados e alongados (IA7 e IA3) apresentaram significativamente maior intensidade de marcação desta que os outros grupos. Não houve diferença estatística das imunomarcações para sulfato de condroitina 4 entre os diferentes grupos. Conclusão: Os protocolos de alongamento muscular, após imobilização, realizados em dias alternados e diariamente, provocaram respostas adaptativas distintas na cartilagem articular. A imobilização desencadeou um quadro de atrofia tecidual que quando estimulada por alongamentos musculares em dias alternados, manteve alguns componentes da matriz, como fibrilas finas de colágeno e proteoglicana. Esta resposta foi agravada quando o mesmo protocolo foi aplicado diariamente. Desta forma, podemos concluir que o alongamento muscular aplicado em articulações previamente imobilizadas deve ser aplicado com cautela, respeitando períodos intercalados de estímulo mecânico.

Made available in DSpace on 2016-06-02T20:18:17Z (GMT). No. of bitstreams: 1 4281.pdf: 12055770 bytes, checksum: 3aea916f9b40945f7cc56297a678e3fd (MD5) Previous issue date: 2012-02-28 / Financiadora de Estudos e Projetos / Objective: To evaluate the chondrocytes apoptosis by caspase 3 expression and its relation with the knee articular cartilage thickness and cellularity, at the acute phase after anterior cruciate ligament (ACL) transection, as the experimental model of osteoarthritis (OA). Material and Methods: Seventy two male Wistars rats were divided into 3 groups: ACL transection group (ACLT); control group (C); and sham group (S). Each group was subdivided into 4 subgroups: 24h, 48h, 72h and 1 week, according to the period following the procedures performed. The medial condiles of the left knees were processed in paraffin and stained with HE for cartilage thickness and cellularity analysis, by morphometry. Caspase 3 expression was evaluated through immunohistochemical method. Statistical analysis was performed by using the Kolmogorov-Smirnov test to assess the normality of the data, and then ANOVA with post hoc Duncan. Results: There was no statistical difference for articular cartilage thickness and cellularity between the groups. Regarding the caspase 3 expression, the C had smaller expression than the other groups. The ACLT 24h and ACLT 48h groups had increased expression of caspase-3 than their respective C and S. The ACLT 72h was not different from S 72h, and presented a lower caspase 3 expression compared to the other groups. The ACLT 7days and S 7days groups had higher expression than any other group. Conclusion: The chondrocyte apoptosis present since the OA beginning, using the ACL transection model of OA, however the articular cartilage thickness and cellularity changes can be modified at a later stage. / Objetivo: Avaliar a presenca de apoptose pela expressao de caspase 3 e sua relacao com a celularidade e espessura do tecido cartilaginoso em joelhos de ratos, na fase aguda apos transeccao do ligamento cruzado anterior (LCA), como modelo experimental de Osteoartrite (OA). Material e Metodos: Setenta e dois ratos Wistars adultos foram divididos em 3 grupos: Grupo Transeccao do LCA (TLCA); Grupo Controle (C); e Grupo Sham (S).Cada grupo foi subdividido em 4 subgrupos: 24hs, 48hs, 72hs e 1 semana, periodo apos os procedimentos realizados. Os joelhos esquerdos dos animais foram utilizados para analise dos condilos femorais. O material foi processado em parafina e corado com HE para a analise da celularidade e espessura da cartilagem por meio de morfometria. Tambem foi realizada analise imunohistoquimica para a expressao de caspase 3. A analise estatistica foi realizada por meio dos testes de Kolmogorov-Smirnov, para avaliar a normalidade dos dados, e posteriormente ANOVA com post hoc de Duncan. Resultados: Nao houve diferenca estatistica para a celularidade e espessura da cartilagem articular entre os grupos estudados. Com relacao a presenca de caspase 3, todos os grupos tiveram maior expressao que os C. Os grupos TLCA 24h e TLCA 48h tiveram maior expressao de caspase-3 que seus respctivos C e S. O TLCA 72h nao foi diferente do S 72h, e apresentou uma menor expressao de caspase 3 com relacao aos outros grupos. O TLCA 7dias e S 7 dias tiveram maior expressaode caspase 3 que todos os outros grupos. Conclusao: A apoptose de condrocito esta presente deste o inicio da OA, quando se usa a transeccao do LCA como modelo experimental, entretanto as mudancas com relacao a celularidade e espessura do tecido poderao se modificar numa fase mais tardia.

Made available in DSpace on 2016-06-02T20:19:25Z (GMT). No. of bitstreams: 1 598.pdf: 612982 bytes, checksum: 1ec87d8ea6133011e61dbd36abec0293 (MD5) Previous issue date: 2005-02-25 / Universidade Federal de Sao Carlos / Objective: The aim of this study is to evaluate the articular cartilage alterations of rat ankles, after applying unilateral cyclic passive muscle stretching protocol for three weeks in previously immobilized rats. Materials and Methods: Twenty-two male albino rats (280+25.4g) divided into four groups: I-immobilized (n=6), IS-immobilized and stretched (n=5), S-stretched (n=6) and C-control (n=5), were used in this experiment. In the immobilization protocol (groups I and IS) the left ankle joints were immobilized in full plantar flexion and this immobilization device was kept on for four weeks. The animal was allowed free cage activity with the device. In the muscle stretching protocol (groups IS and S) the left ankle joint was manually full dorsal flexed 10 times for 60 seconds with a 30s interval between each 60 second period, seven days a week for four weeks, to stretch the ankle plantar flexors muscle group. At the end of the experiment, the ankles were removed, processed in paraffin, cut and stained with Hematoxilin-Eosin and Safranin-O. Two blinded observers graduated histological and histochemical findings related to cellularity, chondrocyte cloning and Safranin-O staining, observed through light microscopy. For histological and histochemical grading, the Mankin et al (1982) and LeRoux et al (2001) modified grading system was used. For statistical analysis it was used the Spearman test, Kruskal-Wallis nonparametric test with the Post Hoc Newman Keuls and Wilcoxon non-parametric test. Results: The previously immobilized stretching group (treated limbs) presented a significantly higher reduction of proteoglycans content than the solely stretched and solely immobilized groups. No significant effect of muscle stretching on treated limbs concerning cellularity and chondrocyte cloning parameters was detected. Conclusion: These findings suggest that the stretching protocol used was harmful to the previously immobilized articular cartilage. However, the same stretching protocol did not harm the cartilage of non-immobilized groups. / Objetivo: Este estudo avaliou a resposta da cartilagem articular do tornozelo de ratos, após aplicação de um protocolo de alongamento muscular passivo cíclico em animais previamente submetidos à imobilização. Materiais e Métodos: Para isso foram utilizados 22 ratos albinos adultos jovens (280+25,4g) distribuídos aleatoriamente em 4 grupos: imobilizado (I), n=6, imobilizado e alongado (IA), n=5, alongado (A), n=6 e controle (C), n=5. Nos grupos I e IA foi utilizado um modelo de imobilização que manteve a pata posterior esquerda imobilizada com o tornozelo, deste membro, em flexão plantar máxima por quatro semanas (pata tratada). Enquanto a pata posterior direita (contralateral) e as anteriores permaneceram livres para o movimento. Após a retirada do aparato de imobilização os grupos IA e A foram submetidos ao protocolo de alongamento muscular por três semanas. Seus tornozelos esquerdos foram manualmente mantidos em flexão dorsal máxima (10 repetições com 60s de duração, intercalados com 30s de relaxamento, sete dias da semana). Após as sete semanas do experimento, os tornozelos foram retirados, processados em parafina, secionados e corados com Hematoxilina-Eosina e Safranina-O. Dois observadores graduaram os achados histológicos e histoquímicos relativos a celularidade, presença de clones e ortocromasia para Safranina-O. As avaliações foram cegas. Para isso foi elaborada uma tabela de pontuação, com nível crescente de lesão, adaptada de dois sistemas de graduação para dano a cartilagem articular descritos na literatura (Mankin et al, 1971 e LeRoux et al, 2001). Os testes estatísticos não paramétricos utilizados foram: Spearman, Kruskal- Wallis com Post Hoc Newman-Keuls e Wilcoxon, considerando grau de significância de 5%. Resultados: As patas tratadas do grupo submetido ao alongamento após a imobilização apresentaram significativamente maior perda do conteúdo de proteoglicanas que as do grupo somente imobilizado. Entretanto o alongamento não gerou alterações teciduais das variáveis estudadas nas patas tratadas dos grupos que não foram previamente imobilizados. Conclusão: Esses dados sugerem que o protocolo de alongamento utilizado nesse estudo foi prejudicial somente à cartilagem articular previamente imobilizada.

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-08-30T19:22:21Z No. of bitstreams: 1 TeseLHGS.pdf: 1716455 bytes, checksum: 7fe0843474a5a5278df1bfaa58e0f403 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-09-25T17:34:48Z (GMT) No. of bitstreams: 1 TeseLHGS.pdf: 1716455 bytes, checksum: 7fe0843474a5a5278df1bfaa58e0f403 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-09-25T17:34:55Z (GMT) No. of bitstreams: 1 TeseLHGS.pdf: 1716455 bytes, checksum: 7fe0843474a5a5278df1bfaa58e0f403 (MD5) / Made available in DSpace on 2018-01-29T17:09:50Z (GMT). No. of bitstreams: 1 TeseLHGS.pdf: 1716455 bytes, checksum: 7fe0843474a5a5278df1bfaa58e0f403 (MD5) Previous issue date: 2017-03-30 / Não recebi financiamento / The aim of the thesis was to present the effects of LLLT, physical exercise and their association on joint cartilage catabolism in the knees of rats submitted to an experimental model of osteoarthritis induced by anterior cruciate ligament (TLCA) transection. For this, a brief systematic review was initially performed where interventions in experimental and clinical studies with animals were considered. The search was performed in the Medline, PubMed, Scopus, Cochrane databases and by active search in a list of bibliographic references of the articles selected until October 2016. The research was carried out according to the guidelines of the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta- Analyzes). We searched the databases with the following terminologies for animal studies: laser therapy and exercise and knee osteoarthritis and animal model. The analyzes of the content of the selected clinical trials demonstrated that LLLT associated therapeutical exercises that can be used to improve pain and quality of life are: resistance exercises and resistance exercises associated with stretching. However, these are not methodologically sufficient to demonstrate improved functionality presenting moderate quality for animal studies and high quality for clinical studies. As the clinical studies included in this systematic review have a high methodological quality, it can be concluded that the therapeutic exercises used by the studies improved pain and functionality in knee OA. However, we suggest experimental studies with animals that may present higher methodological quality to explain the mechanisms of action of therapeutic resources. For this, the second study, investigated the effects of the protocol of resistance exercises and LLLT (associated or not) on the metabolism of cartilage in an experimental model of OA in the knee. Forty male Wistar rats were randomly divided into 4 groups: OA control (OAC); OA and LLLT (OAL); OA and Exercise (OAE); OA and Exercise and Laser (OAEL). The resisted exercise program started 2 weeks after the ACL injury, and were performed three times a week for 8 weeks, totaling 24 sessions consisting of climbing exercises with weights attached to their tails and laser irradiation (two- The results showed that all treated groups had the capacity to modulate some of the aspects related to the degenerative process due to ligament transection, but the association of resistance exercise with LLLT significantly decreased the density of chondrocytes (With or without LLLT) modulated the immunoexpression of IL-1β, caspase-3 and MMP-13. These results suggest that resisted exercises modulate the changes The present work evidences the great need of experimental studies that may explain the clinical mechanisms that favor the use of these resources. In the treatment of patients with OA in the knee. / O objetivo da tese foi apresentar os efeitos da LLLT, exercício físico e a associação dos mesmos sobre o catabolismo na cartilagem articular em joelhos de ratos submetidos a um modelo experimental de osteoartrite induzida através da transecção do ligamento cruzado anterior (TLCA). Para isto, inicialmente foi realizada uma breve revisão sistemática onde foram consideradas intervenções em estudos clínicos. A busca foi realizada nas bases de dados Medline, PubMed, Scopus, Cochrane e por busca ativa em lista de referências bibliográficas dos artigos selecionados de 1994 até outubro de 2016. A pesquisa foi realizada de acordo com as orientações do guia PRISMA (Preferred Reporting Items for Systematic Reviews and Meta- Analyses). Foi realizada a busca nos bancos de dados com as seguintes terminologias: laser therapy and exercise and knee osteoarthritis. As análises do conteúdo dos ensaios clínicos selecionados demonstraram que os exercícios terapêuticos associados à LLLT que podem ser utilizados para melhora da dor e qualidade de vida são: exercícios resistidos e exercícios resistidos associados à alongamentos. Porém, estes não são suficientes metodologicamente para demonstrar melhora da funcionalidade apresentando qualidade moderada para os estudos animais e alta qualidade para os estudos clínicos. Como os estudos clínicos incluídos nesta revisão sistemática têm uma alta qualidade metodológica, pode-se concluir que os exercícios terapêuticos utilizados pelos estudos melhoraram dor e funcionalidade na OA de joelho. Porém sugerem-se estudos experimentais com animais que possam apresentar maior qualidade metodológica para explicar os mecanismos de ação dos recursos terapêuticos. Para isso, o segundo estudo realizado, investigou os efeitos do protocolo de exercícios resistidos e LLLT (associados ou não) no metabolismo da cartilagem em um modelo experimental de OA no joelho. Quarenta ratos Wistar machos foram aleatoriamente divididos em 4 grupos: OA controle (OAC); OA e LLLT (OAL); OA e Exercício (OAE); OA e Exercício e Laser (OAEL). O programa de exercícios resistidos iniciado 2 semanas após a lesão do LCA, e foram realizados três vezes por semana por 8 semanas, totalizando 24 sessões que consistiam de exercícios de escalada, com pesos atados em suas caudas e a irradiação de laser (dois pontos-faces medial e lateral da articulação esquerda. Os resultados mostraram que todos os grupos tratados apresentaram a capacidade de modular alguns dos aspectos relacionados com o processo degenerativo devido à transecção ligamentar. Porém, a associação do exercício resistido com a LLLT diminuiu significativamente a densidade de condrócitos, e em animais OAE houve aumento de densidade. Além disso, os grupos com exercícios resistidos (com ou sem LLLT) modularam a imunoexpressão de IL-1β, caspase-3 e MMP-13. Estes resultados sugerem que os exercícios resistidos modularam as alterações morfológicas relacionadas à progressão da OA. Também, o programa de exercícios apresentou efeitos anti-inflamatórios nos joelhos dos ratos com OA. Com isso, o presente trabalho evidência a grande necessidade de estudos experimentais que possam explicar os mecanismos clínicos que favorecem o uso destes recursos no tratamento de pacientes com OA em joelho.

Abstract Primary hallmark of osteoarthritis (OA) is the progressive degeneration of articular cartilage. An accurate estimation of cartilage mechanics is important when analyzing the subject-specific function of the knee joint and risks for the onset and development of OA due to cartilage damage. Finite element (FE) modeling can help to estimate peak joint stresses and strains and explain how they could lead to OA. FE models of the knee joint during simplified gait were developed to define the level of material complexity required for 3D FE modeling of the knee joint in estimation of reliable tissue stresses and strains within articular cartilage of the knee joint; and to investigate the predictive value of FE modeling of the knee joint on the development and progression of radiographic OA within obese and normal weight subjects. It was found that maximum principal stresses and strains within articular cartilage in the knee joint during walking are highly sensitive to the material parameters of cartilage. It was not possible to match simultaneously stresses, strains and contact pressures between simplified (non-fibrillar) and advanced (fibrillar) models. Though, it was possible to find parameters for transversely isotropic models that enable the estimation of stresses and strains throughout the depth of cartilage similarly to more advanced fibril reinforced models. Locations of peak cumulative stresses in obese subjects at the baseline without radiographic OA showed a good agreement with the locations of cartilage loss and magnetic resonance imaging (MRI) based scoring in four year follow-up when they had developed OA. Simulated weight loss in obese subjects significantly reduced the highest cumulative stresses in cartilage to the level of normal weight subjects. The cartilage degeneration algorithm was able to predict subject-specific progression of OA similarly with MRI follow-up data and separate subjects with radiographic OA from healthy subjects. The computational FE models developed in this thesis represent useful tools to identify possible risk locations within the knee joint and how they relate to OA onset and progression. The presented methods have clinical potential in the diagnostics of knee joint OA in a subject-specific manner and in simulating the effect of interventions on the progression of OA thus helping with an effective treatment planning. / Tiivistelmä Nivelrikon tunnusomaisin piirre on nivelrustokudoksen rappeutuminen ja kuluminen. Nivelruston tehtävänä on tasata niveliin kohdistuvaa kuormitusta. Rustokudoksen mekaanisten ominaisuuksien määrittäminen on tärkeässä roolissa, kun halutaan arvioida tarkemmin polvinivelen toimintakykyä sekä rustokudoksen rappeutumista. Magneettikuvantamisen pohjalta tehtävä polvinivelen biomekaaninen tietokonemallinnus mahdollistaa rustokudoksen jännitys- ja puristusjakauman arvioinnin simuloidun kuormituksen aikana, mikä edelleen voi antaa vastauksia siihen, kehittyykö niveleen tulevaisuudessa nivelrikko, tai miten tietyn nivelrikkopotilaan sairaus etenee. Tämän tutkimuksen päätavoitteena oli kehittää kolmiulotteisia polvinivelen biomekaanisia tietokonemalleja, joiden perusteella simuloitiin normaalia kävelyä. Polvinivelen kolmiulotteinen geometria luotiin terveiden koehenkilöiden sekä nivelrikkopotilaiden magneettikuvista. Malleilla selvitettiin aluksi, miten monimutkaisena materiaalina nivelrusto tulee mallintaa, jotta mallin ennustama jännitys- ja puristusjakauma on silti realistinen. Tämän jälkeen tutkittiin, miten hyvin tietokonemallinnus ennustaa polvinivelrikon kehittymistä ja etenemistä sekä nivelruston rappeutumista ylipainoisilla potilailla. Tutkimuksessa havaittiin, että tietokonemallin ennustamat jännitys- ja puristusjakaumat nivelrustossa kävelyn aikana riippuvat merkittävästi nivelrustolle valitusta materiaalimallista ja sen parametreista. Tietokonemallien ennustamat nivelruston jännityskeskittymien sekä ruston rappeutumisen sijainnit vastasivat erittäin hyvin nivelrustokudoksen todellisen kulumisen sijainteja magneettikuvasta arvioituna neljän vuoden seuranta-ajan jälkeen. Tietokonemalleilla oli myös mahdollista simuloida painon pudotuksen vaikutusta, jolloin nivelrustokudoksen jännitys- ja puristusjakaumat palautuivat normaalien koehenkilöiden tasolle. Tässä tutkimuksessa kehitetyt polvinivelen tietokonemallit tarjoavat tutkijoille uuden työkalun paikallistaa sellaiset kohdat nivelpinnalta, joissa kuormituksen aiheuttama mekaaninen jännitys on suurta; nämä kohdat ovat kaikista riskialtteimpia nivelrikon kehittymiselle. Kehitettyjä malleja voidaan perustutkimuksen lisäksi jatkokehittää edelleen kohti kliinistä sovellusta, jolloin niitä voitaisiin hyödyntää esimerkiksi simuloitaessa erilaisten hoitojen vaikutusta kuormitusjakaumiin ja rustokudoksen rappeutumiseen.