Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Boyan, Barbara; Committee Member: Bonassar, Lawrence; Committee Member: Sambanis, Anthanassios; Committee Member: Schwartz, Zvi; Committee Member: Wick, Timothy.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2007" Includes bibliographical references.

Thesis (Ph. D.)--Bioengineering, Georgia Institute of Technology, 2006. / Harish Radhakrishna, Committee Member ; Christopher Jacobs, Committee Member ; Andres Garcia, Committee Member ; Marc E. Levenston, Committee Chair ; Barbara Boyan, Committee Member.

Habilitation-Thesis--Cologne. / Includes bibliographical references (p. 45-48) and index.

Orientador: Bruno Watanabe Minto / Banca: Luis Gustavo Gosuen Gonçalves Dias / Banca: Paulo César Jark / Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 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. / Mestre

Disorders affecting articular cartilage are amongst the most common problems in orthopaedics. Osteoarthritis, the end stage of the disease of articular cartilage, reduces the quality of life for tens of millions of people in the world, and has a profound impact on the economics of industrialized countries. Despite progress in articular cartilage research, the problem is still far from being defeated. Various models e.g. in vitro cartilage explants and in vivo animal models, have been established for cartilage research, but each has its own limitations. Thus, a novel ex vivo isolated joint organ culture model was developed. Bovine metatarsophalangeal joints were chosen as a suitable synovial joint because it consists of a hinge-type joint that is similar to the human knee joint, and has a large cartilage surface that provides enough space for multiple sampling in the same joint. The joints were isolated aseptically and placed into culture media. The viability of chondrocytes, glycosaminoglycan (GAG) content of cartilage matrix, cartilage morphology and water content of matrix were evaluated under different culture conditions, i.e. static, static with flowing media, and dynamic with different durations of the movement period. The model was used to investigate the effect on the sharp scalpel cartilage injury of adding serum to the culture medium by culturing the whole joint explants in serum-supplied or serum-free media. The feasibility of investigating the early phases of chondrocyte implantation in this model was also studied: circular holes of 2.5 mm diameter were created by making a pilot hole with a 2.0 mm drill followed by using a fresh 2.5 mm biopsy punch. Allogeneic isolated chondrocytes at different passages were aggregated as cell pellets and implanted in the holes to evaluate their integration ability and the response from the recipient cartilage. Results from the static model showed that, after 28 days culture, the chondrocytes were still alive with 66.5%, 80.9% and 46.9% viability in the superficial, middle and deep zones, respectively. The GAG content of the static model decreased 19.2% after the first week of culture and then lost another 15.0% during the third week. Paradoxically, at end of the 4th week the GAG level rebounded to some extent and increased 19.0% relative to the previous week. Interestingly, the cell viability of all three zones improved if the culture fluid was flowing as seen with the experiments carried out with stirred media or dynamic movement of the articular surfaces. (e.g. for the stirred media after 28 days of culture the chondrocyte viability was 80.6%, 92.4% and 70.4% for the superficial, middle and deep zones respectively.) The GAG content was maintained at a constant level in the contact area of the dynamic model, but decreased as in the media-stirred model and non-contact area of the dynamic model to a similar extent to that observed with the static model. In the injury model, the GAG content fell approximately 10.8% straight after the scalpel cut, but no further loss was observed if the joint was cultured in the serum-supplied media. In contrast, if the injured joint was cultured in the serum-free media, the GAG content continued to fall week by week and finally dropped by 41.7% at the end of the 4th week. In the chondrocyte implantation model, the majority of the host chondrocytes around the circular defect were alive (78.5 % viability). Viewed from the surface, the dead cells were all within 20 μm from the cut edge. The implanted chondrocytes, which were aggregated as cell pellets, began to transform their shapes and spread to the surrounding surface of the recipient cartilage, but did not appear to integrate with the host tissue during the first 2 weeks of culture. The results supported the validity of this ex vivo joint model and demonstrated that the chondrocytes subjected to flow of the media or dynamic loads survived well over a 4 week period. Of importance was the finding that there was no measured loss of the matrix GAG content when the joints were under dynamic load compared to all of the non-loaded conditions. This whole joint model could be of value in providing a more natural and controllable platform where research involving the normal processes or pathologic mechanisms of articular cartilage can be investigated, as well as the early response to newly developed pharmacological agents and cartilage tissue engineering constructs.

Orientador: Ibsen Bellini Coimbra / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T22:55:15Z (GMT). No. of bitstreams: 1 Andrade_AndreLuisLugnanide_M.pdf: 2450375 bytes, checksum: f33edb219ba25e56f663f1256a99bbeb (MD5) Previous issue date: 2006 / Resumo: Introdução: Os condrócitos da cartilagem articular vivem em um ambiente com baixa concentração de oxigênio. Nestas condições, a proteína do fator induzido por hipóxia (HIF-1a) mantém-se estável e ativa genes que são fundamentais na homeostase do oxigênio. A expressão do HIF-1a aumenta, em joelhos com osteoartrite (OA), principalmente nas áreas mais afetadas pela degeneração. Os condrócitos são capazes de produzir mediadores inflamatórios, como a interleucina-1 (IL-1) e o fator de necrose tumoral a (TNF-a), que estimulam a produção de prostaglandinas, metaloproteinases e óxido nítrico e relacionam-se com o início e com a progressão da osteoartrite. Os antiinflamatórios são drogas freqüentemente utilizadas no tratamento sintomático da OA. Material e Método: condrócitos humanos de joelhos osteoartríticos cultivados em suspensão e em condições normais de oxigênio foram divididos em quatro grupos: 1) controle, 2) estimulados com IL-1 ou TNF-a, 3) estimulados com meloxicam ou parecoxibe e 4) estimulados com meloxicam ou parecoxibe associados a IL-1 ou TNF-a. Os grupos foram submetidos à extração de RNA (ácido ribonucléico) e de proteína nuclear. O RNA foi convertido em cDNA, sendo então realizada a reação de PCR em tempo real para verificar a expressão do HIF-1a. As proteínas nucleares foram extraídas, quantificadas e analisadas pela técnica de Western Blotting. Resultados: Foi detectada a expressão de HIF-1a e cDNA de HIF-1a em todos os grupos de condrócitos cultivados em suspensão em tensões normais de oxigênio, não havendo diferenças significativas entre os grupos. Discussão: a meia-vida do HIF-1a é extremamente curta em normóxia e marcadamente prolongada em hipóxia, por isso muitos pesquisadores acreditam não ser possível a detecção da proteína do HIF-1a em condrócitos cultivados em condições normais de oxigênio. Neste estudo foi possível constatar a expressão do HIF-1a em normóxia, possivelmente devido ao modelo de cultura utilizado. O estímulo com IL-1, TNF-a e inibidores da COX-2 não alterou a expressão de HIF-1a. Condrócitos oriundos de articulações osteoartríticas avançadas poderiam apresentar resistência à ação das citocinas / Abstract: Introduction: The chondrocytes of joint surface live in low concentration of oxygen environment. In this condition, the hypoxia inducible factor 1 a (HIF-1a) becomes stable and regulates the expression of genes that are important for oxygen homeostasis. The expression of HIF-1a mRNA is augmented in chondrocytes from osteoarthritic knees, especially in more degenerated areas. Chondrocytes are capable of producing inflammatory mediators, such as interleukin 1 (IL-1) and tumoral necrosis factor a (TNF-a), that stimulate the production of prostaglandin, metalloproteinases and nitric oxide, correlated with the onset and progression of osteoarthritis. Antiinflammatory drugs are frequently used in the treatment of symptoms of osteoarthritis. Material e Methods: human chondrocytes from osteoarthritic knees were cultivated in suspension and in normal tension of oxygen. The cells were divided in 4 groups: control, stimulated with IL-1 or TNF-a, stimulated with meloxicam or parecoxib and the last one stimulated with meloxicam or parecoxib and IL-1 or TNF-a. Nuclear protein and RNA were extracted from these cells. cDNA was synthesized from RNA and real time PCR was performed with this product in order to determine HIF-1a expression. Nuclear protein was analyzed using the Western-Blotting method. Results: HIF-1a and HIF-1a mRNA was detectable in all cell groups, and there was not a statistic significant difference between them. Discussion: As half live of HIF-1a is extremely short when in normoxic and greater in hypoxic conditions, many researchers believe it is not possible to detect this protein in chondrocytes cultivated in normoxic environment. Our results presented expression of HIF-1a in normal oxygen tensions, probably due to the fact that chondrocytes were cultivated in suspension. As chondrocytes were obtained from advanced osteoarthritic knees and in such conditions the cells can be more resistant to the action of cytokines, this could explain why IL-1, TNF-a and antiinflamatory did not result in modification of HIF-1a / Mestrado / Clinica Medica / Mestre em Clinica Medica

This thesis deals with the study of the effect of the constituents of the model synovial fluid on the frictional properties of articular cartilage. The influence of constituents, concentration, speed and load is observed. Experiments were performed on a commercial tribometer at configuration pin-on-plate. Several types of lubricants containing synovial fluid constituents have been selected for the experiments. Lubricants were prepared at two concentrations, the concentration of healthy individuals and at a concentration typical of for osteoarthritic patients. Speeds 5 and 10 mm/s and 5 and 10 N loads were used for all experiments. It is shown that when using only lubricant containing proteins, no difference in the coefficient of friction is observed and the effect of concentration is also not observed. The addition of hyaluronic acid has a synergistic effect with -globulin, however in the case of lubricants containing albumin, the effect is opposite. After the addition of phospholipids, no significant effect on friction is observed in -globulin containing lubricants. No significant effect of the composition and concentration of the lubricants is observed with the load change.

Articular cartilage is a highly organized, anisotropic tissue lining the ends of bones within synovial joints. Composed primarily of water, collagens, proteoglycans and chondrocytes which synergistically give rise to the tissue's mechanical and tribological properties. Fluid pressurization and resistance to fluid flow within the porous extracellular matrix of cartilage, coupled with the low hydraulic permeability of the tissue endow the tissue with a viscoelastic response to loading and aid to reduce the coefficient of friction between articulating surfaces, with the pressurized fluid supporting 95% of applied loads. Experiencing millions of articulations throughout an average lifetime, articular cartilage possesses distinct biotribological properties. These require effective lubrication, mediated by the synergistic interaction between fluid and boundary lubricants, to provide a low coefficient of friction and prevent wear at the cartilage surface. Osteoarthritis is the progressive deterioration of articular cartilage and synovial joint structure and function, leading to softer and wear prone tissue on account of altered biochemical composition of the extracellular matrix. Plain radiography remains the most accessible tool and the current standard of care to visualize musculoskeletal diseases and injuries (e.g., osteoarthritis), but cannot directly visualize soft tissues or cartilage, and diagnoses are based solely on boney changes, which occur in the later stages of the disease. Coupled with no way to quantitatively assess tissue health prior to irreversible deterioration, there remains no cure for osteoarthritis. Integral to OA pathology are concomitant changes in the biochemical composition of synovial fluid that result in deterioration of rheological properties, contributing to increased cartilage wear. To address both the lack of quantitative diagnosis methods and lack of chondroprotective therapies, this dissertation presents a dual faceted approach to quantitatively image articular cartilage health, coupled with lubrication strategies to improve cartilage lubrication, and preserve cartilage tissue. This dissertation describes the synthesis of tantalum oxide nanoparticles of varying surface charges for use as contrast agents for rapid, minimally invasive, non-destructive, and quantitative contrast-enhanced computed tomography to assess both the biochemical content and biomechanical integrity of articular cartilage. Ex vivo contrast enhanced computed tomography attenuation using the nanoparticle contrast agent reveals correlations between attenuation and the mechanical and biochemical properties of the tissue. The lubrication strategy described within this dissertation involves introducing a rolling ball element between two surfaces to reduce friction. In this strategy, either single, globular macromolecules or nanoparticles are employed as ball bearings between articulating surfaces to reduce friction when asperities on the surfaces are in direct contact. Rheological characterization and construction of classical Stribeck curves using the lubricant formulations reveal that introducing the rolling element reduces the coefficient of friction during boundary lubrication, while leaving the rheological properties of the base fluid intact. Ex vivo cartilage mechanical testing involving shear deformation under varying speeds and loads reveal improved biotribological performance compared to pure synovial fluid or saline.

First, an in vitro growth experiment was performed to test the hypothesis that applying dynamic unconfined compression during culture produces het- erogeneous remodeling in newborn bovine articular cartilage explants. Het- erogeneous measures of cartilage microstructure were obtained by biochemical assays and quantified polarized light microscopy. Significant differences were measured between the GAG content in the inner and outer portions of the sam- ples stimulated with dynamic unconfined compression. The COL fiber network was found to be more highly aligned in the inner portion of the sample than in the peripheral region. Next, a poroelastic finite element model with a remodeling subroutine was developed to test the hypothesis that the magnitude of relative interstitial fluid velocity and maximum principle strain stimulate GAG and COL fiber network remodeling, respectively, in articular cartilage during culture with dynamic unconfined compression. The GAG remodeling law was successful in predicting the heterogeneous changes in GAG content. The collagen remodeling law was not successful in predicting the changes in the COL network microstructural orientation, suggesting another mechanical cue is responsible for stimulating the remodeling of the COL fiber network.