The development of bioengineered cartilage for replacement of damaged articular cartilage has gained momentum in recent years. One such approach has been developed in the Kandel lab, whereby cartilage is formed by seeding primary articular chondrocytes on the top surface of a porous biodegradable calcium polyphosphate (CPP) bone substitute, permitting anchorage of the tissue within the pores of the substrate; however, the interfacial shear properties of the tissue-substrate interface of these biphasic constructs are 1 to 2 orders of magnitude lower than the native cartilage-subchondral bone interface. To overcome this limitation, a strategy was devised to generate a zone of calcified cartilage (ZCC), thereby mimicking the native architecture of the osteochondral junction; however, the ZCC was located slightly above the cartilage-CPP interface. Thus, it was hypothesized that polyphosphate released from the CPP substrate and accumulating in the tissue inhibits the formation of the ZCC at the tissue-substrate interface. Based on this information, a strategy was devised to generate biphasic constructs incorporating a properly located ZCC. This approach involved the application of a thin calcium phosphate film to the surfaces of porous CPP via a sol-gel procedure, thereby limiting the accumulation of polyphosphate in the cartilaginous tissue. This modification to the substrate surface did not negatively impact the quality of the in vitro-formed cartilage tissue or the ZCC. Interfacial shear testing of biphasic constructs demonstrated significantly improved interfacial shear properties in the presence of a properly located ZCC. These studies also led to the observation that chondrocytes produce endogenous polyphosphate and that its levels in deep zone cartilage appear inversely related to mineral deposition within the tissue. Using an in vitro model of cartilage calcification, it was demonstrated that polyphosphate levels are modulated in part by the inhibitory effects of fibroblast growth factor 18 on exopolyphosphatase activity in the tissue. Polyphosphate also appears to act in a feedback loop to control exopolyphosphatase activity. Interestingly, polyphosphate also exhibits positive effects on cartilage matrix accumulation. The potential implication of polyphosphate in the maintenance of articular cartilage homeostasis is intriguing and must be investigated further.

The development of bioengineered cartilage for replacement of damaged articular cartilage has gained momentum in recent years. One such approach has been developed in the Kandel lab, whereby cartilage is formed by seeding primary articular chondrocytes on the top surface of a porous biodegradable calcium polyphosphate (CPP) bone substitute, permitting anchorage of the tissue within the pores of the substrate; however, the interfacial shear properties of the tissue-substrate interface of these biphasic constructs are 1 to 2 orders of magnitude lower than the native cartilage-subchondral bone interface. To overcome this limitation, a strategy was devised to generate a zone of calcified cartilage (ZCC), thereby mimicking the native architecture of the osteochondral junction; however, the ZCC was located slightly above the cartilage-CPP interface. Thus, it was hypothesized that polyphosphate released from the CPP substrate and accumulating in the tissue inhibits the formation of the ZCC at the tissue-substrate interface. Based on this information, a strategy was devised to generate biphasic constructs incorporating a properly located ZCC. This approach involved the application of a thin calcium phosphate film to the surfaces of porous CPP via a sol-gel procedure, thereby limiting the accumulation of polyphosphate in the cartilaginous tissue. This modification to the substrate surface did not negatively impact the quality of the in vitro-formed cartilage tissue or the ZCC. Interfacial shear testing of biphasic constructs demonstrated significantly improved interfacial shear properties in the presence of a properly located ZCC. These studies also led to the observation that chondrocytes produce endogenous polyphosphate and that its levels in deep zone cartilage appear inversely related to mineral deposition within the tissue. Using an in vitro model of cartilage calcification, it was demonstrated that polyphosphate levels are modulated in part by the inhibitory effects of fibroblast growth factor 18 on exopolyphosphatase activity in the tissue. Polyphosphate also appears to act in a feedback loop to control exopolyphosphatase activity. Interestingly, polyphosphate also exhibits positive effects on cartilage matrix accumulation. The potential implication of polyphosphate in the maintenance of articular cartilage homeostasis is intriguing and must be investigated further.

Articular cartilage on weight-bearing joints experiences three main forces: fluid-induced shear across the surface, perfusion through the cartilage from the surrounding fluid, and compression during motion of the joint. A new bioreactor that employs two of these forces was developed in this lab to study their effect on tissue-engineered cartilage development. The focus of this research and overall hypothesis is that bioreactors that employ both perfusion and shear will improve chondrogenesis and preservation to produce functionally relevant cartilage by modulating shear stress and introducing exogenous preservation factors. Applying both a low shear stress across the surface of cell-seeded scaffolds and perfusion through them in a perfusion concentric cylinder (PCC) bioreactor may stimulate chondrocytes to undergo chondrogenesis. Experimental data showed that the PCC bioreactor stimulated cartilage growth over the course of four weeks, supported by the appearance of glycosaminoglycan (GAG) and collagen type II, which are markers for articular cartilage. Computational fluid dynamics modeling showed that shear stress across the face of the construct was heterogeneous, and that only the center experienced a relatively uniform shear stress of 0.4 dynes/cm^2 when the outer cup of the bioreactor rotated at 38 rpm. When compared to a concentric cylinder (CC) bioreactor that employed only shear stress, the PCC bioreactor caused a significant increase in cellular proliferation, which resulted in a 12-fold increase in cell number per construct compared to 7-fold increase within the CC bioreactor. However, the PCC bioreactor had a less pronounced effect on glycosaminoglycan and collagen content with 1.3 mg of GAG and 1.8 mg of collagen per construct within the CC bioreactor and 0.7 mg of GAG and 0.8 mg of collagen per construct within the PCC bioreactor after 28 days in culture (p < 0.05). Our results led to an important observation that the PCC bioreactor affected cellular proliferation significantly but not extracellular matrix synthesis. The next objective of this study focused on the PCC bioreactor to evaluate the direct role of perfusion and shear on chondrogenesis in vitro and in vivo.

Osteoarthritis is a degenerative joint disease characterized by progressive erosion of the articular cartilage. Epidemiological studies have established a relationship between osteoarthritis and menopause suggesting that estrogen may be important in the development of cartilage regeneration therapies. The overall goal of this research project was to advance the field of cartilage tissue regeneration by investigating the role of 17 ß -estradiol (E2), an active estrogen metabolite, on the chondrocyte phenotype. The central hypothesis was that E2 plays an important and sex-specific role in regulating chondrogenesis. Specific Aim-1 focused on establishing and characterizing a primary human articular chondrocyte (HAC) cell source, and then examining the response of the cells in culture to E2. It was demonstrated that the response of HACs to E2 treatment was sex-specific despite both male and females cells expressing estrogen receptors. Female HACs showed changes in proliferation, matrix production, and differentiation while male cells did not. In addition, the female response was regulated through a rapid membrane signaling pathway mediated by protein kinase C. Specific Aim-2 involved establishing an ovariectomized animal model to investigate the effects of E2 on orthopaedic tissue implants. Human demineralized bone matrix (DBM) was implanted intramuscularly into female nude mice and rats. Ovariectomy was shown to reduce the ability of DBM to induce the formation of cartilage and bone tissue. Moreover, the inductive properties of DBM were reestablished with subcutaneous E2 supplementation. Specific Aim-3 entailed developing and characterizing a microencapsulation method for in vitro culture and in vivo delivery of chondrocytes to study the effects of E2 on chondrogenesis. Rat growth plate chondrocytes and HACs were microencapsulated in alginate using an extrusion method in conjunction with high electrostatic potential. Chondrocytes maintained their phenotype in alginate suspension but were unable to form cartilage tissue when implanted into our animal model. Further optimization of the system is required before the role of E2 on chondrogenesis of tissue engineered constructs can be determined. In summary, our results suggest that the successful production of tissue engineered therapies will likely depend on understanding and manipulating the actions of sex hormones in both the in vitro and in vivo environment.

[Truncated abstract] Hyaline articular cartilage is a highly specialised tissue consisting of chondrocytes embedded in a matrix of proteoglycan and collagens. Hyaline articular cartilage withstands high levels of mechanical stress and continuously renews its extracellular matrix. Despite this durability, mature articular cartilage is vulnerable to injury and disease processes that cause irreparable tissue damage. Native hyaline articular cartilage has poor regenerative capacity following injury, largely due to the tissue's lack of blood and lymphatic supply, as well as the inability of native chondrocytes to migrate through the dense extracellular matrix into the defect site. Articular cartilage injuries that fail to penetrate the subchondral bone plate evoke only a short-lived metabolic and enzymatic response, which fails to provide sufficient new cells or matrix to repair even minimal damage. Clinically, it has previously been accepted that treatment of such defects does not result in the restoration of normal hyaline articular cartilage, which is able to withstand the mechanical demands that are placed on the joint during every day activities of daily living. ... Historically, rehabilitation following ACI has not kept pace with the advances in cell culture and surgical technique. Subsequently, there exists a significant gap in knowledge regarding `best practice' in post operative rehabilitation following ACI. The importance of structured rehabilitation in ACI should not be underestimated when evaluating the clinical success of this chondral treatment. Patients should not be left to their own devices following ACI surgery, as the risk of damage to their implant (via delamination) is high if immediate postoperative movement is not controlled. Furthermore, the biological longevity and clinical success of the graft is dependent on a controlled and graduated return to ambulation and physical activity, and the biomechanical stimulation of the implanted chondrocytes.

Made available in DSpace on 2016-06-02T19:02:43Z (GMT). No. of bitstreams: 1 5751.pdf: 1870522 bytes, checksum: c62c3a70629b608363bff844d45283ba (MD5) Previous issue date: 2013-02-28 / Universidade Federal de Sao Carlos / Osteoarthritis (OA) is characterized as a chronic disease that affects synovial joints, causing degeneration and inflammation. Treatments for OA include painkillers and antiinflammatory drugs, physical exercises and, in the most serious cases, surgical interventions. Recently, can be emphasized the use of non-invasive technologies such as low level laser therapy (LLLT). In this context, the aim of this study was to evaluate the effects of LLLT on the metabolism of articular cartilage on the knees of rats in an experimental model of OA. Eighty-male rats (Wistar) were distributed into four groups: intact group (GI); injured control group (GC); injured laser treated group at 10J/cm2 (L10) and injured laser treated group at 50J/cm2 (L50). Animals were distributed into 2 subgroups, with different periods of sacrifice (5 and 8 weeks post-surgery). The anterior cruciate ligament transection (ACLT) was used to induce OA in the knee of rats. The LLLT (&#955;=830nm, 10 and 50J/cm2) started 2 weeks after the surgery and it was performed for 15 and 30 sessions. Qualitative and semi-quantitative histology, morphometry, immunohistochemistry and enzyme-linked immunosorbent Assay (ELISA) analysis were performed. The histological findings revealed that laser therapy modulated the progression of the degenerative process. However, laser therapy was not able of decreasing the degenerative process observed by the increased of Mankin score and cartilage thickness and it did not have any effect in the biomodulation of the expression of markers IL1&#946;, TNF-&#945; and MMP-13. Moreover, LLLT, at 50J/cm2 was able to increase the expression of cytokines IL-4 and IL-10. The results found in present study indicate that laser therapy 830 nm, modulate proliferation of chondrocytes in the experimental model of OA in rats but had no effect on the expression of cytokines IL-1&#946; and TNF-&#945;. The laser therapy 50J/cm2 LLLT was able in increasing serum expression of cytokines IL-4 and IL-10, favoring a regulatory effect of the inflammatory process. / A osteoartrite (OA) é caracterizada como uma doença crônica que afeta as articulações sinoviais causando degeneração e inflamação. O tratamento da OA consiste em drogas analgésicas e anti-inflamatórias, exercícios físicos e, em casos mais graves, intervenção cirúrgica. Mais recentemente, pode ser destacado o uso de tecnologias não invasivas como a terapia laser de baixa intensidade (LLLT). Com isso, o objetivo deste estudo foi avaliar os efeitos da LLLT no metabolismo da cartilagem articular do joelho de ratos submetidos a um modelo experimental de OA. Um total de 80 ratos machos (Wistar) foram distribuídos aleatoriamente em 4 grupos: grupo intacto (GI); grupo controle lesão (GC); grupo lesão tratado com laser na fluência de 10J/cm2 (L10) e grupo lesão tratado com laser na fluência de 50J/cm2 (L50). Os animais foram distribuídos em 2 subgrupos, com diferentes períodos de sacrifício (5 e 8 semanas pós-cirurgia). A transecção do ligamento cruzado anterior (TLCA) foi utilizada para induzir a OA no joelho dos ratos. O tratamento com laser de baixa intensidade (&#955;= 830nm, 10 e 50J/cm2) teve início 2 semanas após a cirurgia e foi realizado em 15 ou 30 sessões. Para avaliar os efeitos do laser no processo osteoartrítico, foram realizadas análises histológicas qualitativas e semi-quantitativas, morfometria, imunohistoquimica e ensaio imunoenzimático. Os resultados histológicos revelaram que a terapia laser parece modular a progressão do processo de degeneração da cartilagem articular. Porém, o laser não foi capaz de diminuir o processo degenerativo observado através do aumento na graduação de Mankin e da espessura da cartilagem e ainda não apresentou efeito sobre a biomodulação da expressão das citocinas IL-1&#946;, TNF-&#945; e MMP-13. No entanto, a terapia laser na fluência de 50J/cm2 foi capaz de aumentar a expressão das citocinas regulatórias (IL-4 e IL-10). Deste modo, os resultados do presente estudo indicam que a terapia laser 830nm modula a proliferação de condrócitos em modelo experimental de OA em ratos, mas não apresenta efeito na expressão das citocinas IL-1&#946; e TNF-&#945;. A terapia laser 50J/cm2 foi capaz de aumentar a expressão sérica das citocinas IL-4 e IL- 10, favorecendo um efeito regulatório do processo inflamatório.

Made available in DSpace on 2015-03-26T13:46:49Z (GMT). No. of bitstreams: 1 texto completo.pdf: 877851 bytes, checksum: e19d9e07d366a220755f330c28b830fe (MD5) Previous issue date: 2005-04-15 / The aim of this study was to evaluate the clinical and morphophisiological aspects of the rat knee articulation after immobilization and remobilization with free activity and swimming. Thirty four adult male rats (Wistar) were randomly allocated into one of four groups: without immobilization, (G1, n = 4), with immobilization (G2, n = 10), immobilization and remobilization with free activity (G3, n = 10), and immobilization and remobilization with free activity and swimming (G4, n = 10). Animals had the right knee joint immobilized for 45 days. Animals from G2 were killed after this immobilization period and so were those from G1. After the immobilization period animals from G3 were allowed to move freely in their cages and those from G4 add to that were submitted to a swimming program for 5 weeks and were than all killed. During the remobilization period animals from G3 and G4 had their march clinically evaluated. After being killed the articular amplitude of the right knee was assessed in all animals and histological slices from the articular capsule, cartilage and subchondral bone were obtained. The results showed that animals from G4 exhibited a better march evolution on the first five days period as compared to those from G3. After such period march evolution were similar in both G3 and G4. Animals from G2 displayed rigid joint while in those from G3 and G4 the articular movement was close to normal. The histological analysis pointed out that immobilization led to increase in articular capsule thickness evidenced by the presence of fibrous connective tissue replacing adipose tissue in G2, but proportionally less in G3 and G4. By the analysis of the articular cartilage and subchondral bone it was observed that immobilization determined loss of proteoglycans from the cartilaginous matrix, increase in the number of regularly arranged condrocytes, increase in calcified cartilage thickness, irregularity in the articular surface, proliferation of connective tissue in the intraarticular space and increase in subchondral bone thickness. A animals from G3 displayed a greater number of alterations in both articular cartilage and subchondral bone, as compared to those from G4. It was concluded that: immobilization degenerated synovial cells indicating decreased synovial fluid production and reduced nutritional supplying to the cartilage; and both free cage activity and its association with swimming influenced positively the return of the capsule morphologic conditions to those before immobilization. / O objetivo do presente trabalho foi avaliar os aspectos clínicos e morfofisiológicos da articulação do joelho de ratos após imobilização prolongada e remobilização com atividade livre e natação. Trinta e quatro ratos adultos machos (Wistar) foram alocados aleatoriamente em um dos quatro grupos: sem imobilização (G1, n = 4), com imobilização (G2, n = 10), imobilização e remobilização com atividade livre (G3, n = 10), imobilização e remobilização com atividade livre e natação (G4, n = 10). Os animais tiveram o joelho direito imobilizado por 45 dias. Os animais do G2 foram sacrificados após o período de imobilização, juntamente com os animais do G1. Após o período de imobilização os animais do G3 movimentaram livremente em suas respectivas gaiolas e os animais do G4, além da movimentação na gaiola, foram submetidos a um programa de natação por 5 semanas, sendo todos sacrificados em seguida. Durante este período de remobilização os animais dos G3 e G4 foram submetidos a avaliação clínica da marcha. Após sacrifício a resistência à flexão articular do joelho direito de todos os animais foi avaliada e cortes histológicos da cápsula articular, cartilagem e osso subcondral foram obtidos. Os resultados mostraram que os animais do G4 apresentaram melhor evolução na marcha nos cinco primeiros dias, em relação àqueles do G3. Após este período a evolução na marcha foi similar nos G3 e G4. Animais do G2 apresentaram rigidez articular enquanto nos do G3 e G4 o movimento estava próximo ao normal. A análise histológica mostrou que a imobilização promoveu aumento da espessura da cápsula articular, evidenciada pela presença do tecido conjuntivo fibroso substituindo o tecido adiposo no G2, mas em menor proporção nos G3 e G4. Pela análise da cartilagem articular e osso subcondral pode-se observar que a imobilização determinou perda de proteoglicanos da matriz cartilaginosa, aumento do número de condrócitos dispostos de forma irregular, aumento da espessura da cartilagem calcificada, irregularidade da superfície articular, proliferação de tecido conjuntivo no espaço intra-articular e aumento da espessura do osso subcondral. Os animais do G3 apresentaram um maior número de alterações tanto na cartilagem articular como no osso subcondral, quando comparados com aqueles do G4. Concluiu-se que a imobilização degenerou as células sinoviais, indicando diminuição da produção de fluido sinovial e redução do suprimento nutricional à cartilagem; e que tanto a atividade livre na gaiola quanto sua associação com a natação influenciaram positivamente o retorno das condições morfológicas da cápsula, anteriores à imobilização.

Submitted by LÍVIA DE PAULA COELHO null (coelholivia@hotmail.com) on 2017-04-25T16:42:01Z No. of bitstreams: 1 Dissertação_Lívia_de Paula_Coelho.pdf: 1644637 bytes, checksum: 23b5d8d8d626124c1f92f58da2470387 (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido não contém o certificado de aprovação. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-04-26T16:31:22Z (GMT) / Submitted by LÍVIA DE PAULA COELHO null (coelholivia@hotmail.com) on 2017-04-28T15:28:35Z No. of bitstreams: 1 Dissertação_Lívia_de Paula_Coelho.pdf: 1693903 bytes, checksum: 19b7ed665d5907083e027dbd119748c8 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-05-03T13:39:08Z (GMT) No. of bitstreams: 1 coelho_lp_me_jabo.pdf: 1693903 bytes, checksum: 19b7ed665d5907083e027dbd119748c8 (MD5) / Made available in DSpace on 2017-05-03T13:39:08Z (GMT). No. of bitstreams: 1 coelho_lp_me_jabo.pdf: 1693903 bytes, checksum: 19b7ed665d5907083e027dbd119748c8 (MD5) Previous issue date: 2017-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A cartilagem articular possui capacidade de reparação limitada, aumentado a predisposição ao desenvolvimento de alterações degenerativas, muitas vezes irreversíveis. Diversas formas de tratamento, cirúrgicas ou conservativas, são descritas, entretanto a terapêutica da osteoartrite continua sendo grande desafio ao médico veterinário. Neste contexto, a pesquisa envolvendo células-tronco mesenquimais destaca-se na busca de melhorias e avanços na reparação da cartilagem articular. Objetivou-se, no presente projeto, comparar a regeneração cartilaginosa da articulação coxofemoral de coelhos, com e sem o transplante de células-tronco mesenquimais autólogas, por meio de exames radiográficos e histopatológicos. Dois grupos, com 15 animais da espécie leporina cada, foram submetidos à indução química de osteoartrite com solução de colagenase 2% na articulação coxofemoral direita. No Grupo 1 (Células-tronco) realizou-se a aplicação intra-articular de células-tronco mesenquimais autólogas, enquanto que, o Grupo 2 (Controle) foi constituído por animais submetidos à aplicação intra-articular de solução salina estéril. Foram realizadas avaliações radiográficas e histopatológicas aos 30, 60 e 90 dias após a aplicação. Os resultados histológicos deste ensaio indicam que células-tronco mesenquimais (Grupo 1) melhoraram discretamente a qualidade do tecido de reparo, de acordo com os critérios da escala semi-quantitativa ICRS 1 (“International Cartilage Repair Society”). O Grupo 1 (Células-Tronco) demonstrou superioridade em relação ao Grupo 2 principalmente nos parâmetros: Superfície articular, matriz extracelular e distribuição celular. / The articular cartilage has limited repair capacity, leading to an increased risk for degenerative changes, potentially irreversible. Several treatments, surgical or not, are described, however osteoarthritis remains a major challenge for the veterinarian. In this context, research involving mesenchymal stem cells stands out. The aim of this study was to compare cartilage regeneration of the hip in rabbits, with and without the transplantation of autologous mesenchymal stem cells. Radiographic and histopathological evaluation were used. Thirty rabbits were submitted to chemical induction of osteoarthritis with a 2% colagenase in the right hip. They were divided into 2 groups of 15 animals each: Group 1 (intra-articular application of autologous mesenchymal stem cells) and Group 2 (control - intra-articular application of sterile saline solution). Radiographic and histopathological evaluations were performed at 30, 60 and 90 days after application. The mesenchymal stem cells group (Group 1) showed slight improvement of the quality of the repair tissue, according to the semi-quantitative scale criteria ICRS 1 (International Cartilage Repair Society). The Group 1 (Stem Cells) showed superiority in relation to Group 2, specially in the parameters joint surface, extracellular matrix and cellular distribution.

Abstract Cartilage-related diseases such as osteoarthritis (OA) are a major cause of disability and decrease in the quality of life. Moreover OA causes a heavy economical burden on the social welfare and health care systems. Conventional magnetic resonance imaging (MRI) provides accurate noninvasive method of morphological evaluation of the articular cartilage. However, there are early degenerative changes in the articular cartilage that can be evaluated with modern quantitative MRI methods prior to the signs of cartilage loss. In this study, T2 relaxation time of the articular cartilage was further evaluated in 1.5T in vivo using clinical patients and asymptomatic volunteers. The detection of focal patellar cartilage lesions in T2 mapping as compared to standard clinical MRI was evaluated. T2 mapping showed more lesions than the clinical MRI, and in T2 maps the lesions appeared generally wider. This suggests that T2-mapping is feasible in the clinical setting and may reveal cartilage lesions not seen in the standard knee MRI. The normal topographical variation of T2 relaxation time of articular cartilage in different compartments of the knee joint and at different zones of cartilage in young healthy adults was assessed. T2 values were significantly higher in the superficial zone as compared to the deep tissue at all locations and there was remarkable variation in T2 relaxation between different locations. The normal variation in cartilage T2 within a joint is significant and should be acknowledged when pathology-related T2 changes are investigated. The short- and long-term repeatability of T2 relaxation time measurements of articular cartilage in the knee joint was assessed. The results showed mostly good repeatability, and with careful patient positioning T2 relaxation time at the different cartilage surfaces of the knee can be accurately determined. / Tiivistelmä Nivelrikko, joka usein liittyy nivelruston vaurioitumiseen, aiheuttaa merkittävää toimintakyvyn ja elämänlaadun heikentymistä ikääntyvässä väestössä. Lisäksi nivelrikosta aiheutuu merkittäviä kustannuksia sosiaali- ja terveydenhuollolle. Magneettikuvaus on tarkka kajoamaton menetelmä rustovaurioiden arvioimiseksi. Kuitenkin rustovaurion alkuvaiheessa tapahtuu ruston sisäisiä rakenteellisia ja biokemiallisia muutoksia, joita on mahdollista arvioida uusilla kvantitatiivisilla magneettikuvausmenetelmillä ennen varsinaisten rustopuutosten kehittymistä. Tässä tutkimuksessa tutkittiin ruston T2-relaksaatioaikamittausta 1.5T magneettikuvauslaitteella sekä potilasaineistossa että vapaaehtoisilla. Tutkimuksessa verrattiin paikallisten rustomuutosten havaitsemisen herk¬kyyttä T2-relaksaatioaikakartoituksen ja tavanomaisen kliinisen magneetti¬kuvauksen välillä kliinisessä potilasaineistossa. T2-relaksaatiomittaus osoitti useampia muutoksia kuin kliininen magneettikuvaus ja muutokset olivat yleensä laajempia. Voidaan olettaa, että T2-relaksaatioaikamittaus soveltuu kliiniseen käyttöön ja voi osoittaa tavanomaisessa magneettikuvauksessa näkymättömiä rustomuutoksia. Tutkimuksessa arvioitiin ruston T2-relaksaatioajan paikkakohtaista ja kerroksittaista vaihtelua polven nivelpintojen eri alueilla nuorten vapaaehtoisten aineistossa. T2-relaksaatioaika oli merkitsevästi pidempi ruston pinnallisessa kuin syvässä kerroksessa kaikilla nivelpintojen alueilla. Lisäksi T2-relaksaatioajassa oli merkittävää normaalia vaihtelua eri alueiden välillä ja tämä tulisi huomioida ruston patologisia muutoksia arvioitaessa. Tutkimuksessa arvioitiin polven ruston T2-relaksaatioajan lyhyen ja pitkän aikavälin toistettavuutta vapaaehtoisaineistossa. Tulokset osoittivat enimmäkseen hyvää toistettavuutta ja huolellisella asettelulla voidaan ruston T2-relaksaatioaika mitata luotettavasti polven nivelpintojen eri alueilla.

The Runx family of transcription factors supports cell fate determination, cell cycle regulation, global protein synthesis control, and genetic as well as epigenetic regulation of target genes. Runx1, which is essential for hematopoiesis; Runx2, which is required for osteoblast differentiation; and Runx3, which is involved in neurologic and gut development; are expressed in the growth plate during chondrocyte maturation, and in the chondrocytes of permanent cartilage structures. While Runx2 is known to control genes that contribute to chondrocyte hypertrophy, the functions of Runx1 and Runx3 during chondrogenesis and in cartilage tissue have been less well studied. The goals of this project were to characterize expression of Runx proteins in articular cartilage and differentiating chondrocytes and to determine the contribution of Runx1 to osteoarthritis (OA). Here, the expression pattern of Runx1 and Runx2 was characterized in normal bovine articular cartilage. Runx2 is expressed at higher levels in deep zone chondrocytes, while Runx1 is primarily expressed in superficial zone chondrocytes, which is the single cell layer that lines the surface of articular cartilage. Based on this finding, the hypothesis was tested that Runx1 is involved in osteoarthritis, which is a disease characterized by degradation of articular cartilage and changes in chondrocytes. These studies showed that Runx1 is upregulated in articular cartilage explants in response to mechanical compression. Runx1 was also expressed in chondrocytes found at the periphery of OA lesions in the articular cartilage of mice that underwent an OA-inducing surgery. Runx1 was also upregulated in cartilage explants of human osteoarthritic knees, and IHC data showed that Runx1 is mainly expressed in chondrocyte “clones” characteristic of OA. To ascertain the potential function of the upregulation of Runx1 in these cartilage stress conditions and disease states, the hypothesis was tested that Runx1 is upregulated in very specific chondrocyte populations in response to the cartilage damage in osteoarthritis. These studies addressed the properties of these cells that related to functions in cell growth and differentiation. In both the surface layer of normal articular cartilage, and in OA cartilage, Runx1 expression by IF co-localized with markers of mesenchymal progenitor cells, as well as markers of proliferation Ki-67 and PCNA. This finding indicated that Runx1 is found in a population of cells that represent a proliferative population of mesenchymal progenitor cells in osteoarthritis. To further address Runx1 function and identify downstream targets of Runx proteins, a promoter analysis of genes that are known to be either downregulated or upregulated during chondrocyte maturation was done. These studies found that many of these genes have 1 or more Runx binding sites within 2kb of their transcription start site, indicating that they are potential downstream Runx target genes. Lastly, some preliminary experiments were done to characterize novel roles of Runx proteins in the chondrocyte. Runx proteins have been shown to epigenetically regulate their target genes by remaining bound to them throughout mitosis, “poising” them for transcription upon exit from mitosis. The hypothesis that Runx proteins also function by remaining bound to their target genes throughout mitosis in chondrocytes was tested. It was demonstrated by immunofluorescense imaging of Runx proteins on metaphase chromosomes of ATDC5 cells, that Runx2 remains bound to chromosomes during mitosis. Cell proliferation and hypertrophy are both linked to increases in protein synthesis. Runx factors, which regulate rates of global protein synthesis, are expressed in both proliferating and hypertrophic chondrocytes. Thus, it was hypothesized that Runx proteins regulate rates of global protein synthesis during chondrocyte maturation. These studies showed that the overexpression of Runx proteins in a chondrocyte cell line (ATDC5) did not affect protein synthesis rates or levels of protein synthesis machinery. Additionally, Runx proteins did not affect proliferation rates in this chondrocyte cell line.