<p>The role of articular cartilage in diarthrodial joints is primarily mechanical as the tissue provides a nearly frictionless, load-bearing surface that supports and distributes forces generated during joint loading. Embedded within the extensive cartilage extracellular matrix (ECM), chondrocytes are surrounded by a narrow, distinct pericellular matrix (PCM) that is thought to regulate the biomechanical microenvironment of the cell and influence chondrocyte metabolism, cartilage homeostasis, and overall joint health. While previous studies of PCM mechanical properties required physical extraction of the cell and PCM from the tissue, atomic force microscopy (AFM) provides a means for high resolution microindentation testing that can be used to measure local mechanical properties in situ. This dissertation develops and applies AFM microindentation techniques to 1) evaluate the microscale elastic properties of the cartilage PCM and ECM in situ and 2) correlate site-specific biochemical composition with biomechanical properties of the PCM and ECM. </p><p>An AFM-based stiffness mapping technique was experimentally validated and applied to cartilage sections to evaluate ECM and PCM properties in situ with minimal disruption of native matrix integration. As expected, PCM elastic moduli were significantly less than ECM moduli, uniform with depth, and mechanically isotropic. ECM moduli exhibited distinct depth-dependent anisotropy and unexpectedly, were found to decrease with depth from the articular surface. Both the PCM and ECM exhibited alterations in microscale moduli and their spatial distributions when evaluated in cartilage presenting early degenerative changes associated with osteoarthritis (OA) as compared to healthy tissue. </p><p>The ability to correlate site-specific biochemical composition with local biomechanical properties provides a more complete characterization of the chondrocyte microenvironment. To this end, we developed novel immunofluorescence (IF)-guided AFM stiffness mapping and demonstrated that PCM mechanical properties correlate with the presence of type VI collagen. Extending this technique by using dual IF, we presented new evidence for a defining role of perlecan in the PCM, showing that interior regions of the PCM rich in perlecan and type VI collagen exhibit lower elastic moduli than peripheral PCM and ECM regions lacking perlecan. Furthermore, lower moduli at the PCM interior were significantly influenced by the presence of heparan sulfate. IF-guided AFM stiffness mapping was combined with enzymatic digestion to demonstrate that the micromechanical properties of the PCM exhibit high resistance to specific enzymatic digestion of aggrecan and aggrecan-associated glycosaminoglycans but are vulnerable to proteolytic degradation by leukocyte elastase. </p><p>Overall, this research generates new insights into the complex structural, compositional, and functional relationships between the cartilage ECM and PCM and provides the tools and framework for further studies to continue to investigate their importance in regulating chondrocyte physiology in health and disease.</p> / Dissertation

In Alzheimer’s disease (AD), different types of neurons and different brain areas show differential patterns of vulnerability towards neurofibrillary degeneration, which provides the basis for a highly predictive profile of disease progression throughout the brain that now is widely accepted for neuropathological staging. In previous studies we could demonstrate that in AD cortical and subcortical neurons are constantly less frequently affected by neurofibrillary degeneration if they are enwrapped by a specialized form of the hyaluronan-based extracellular matrix (ECM), the so called ‘perineuronal net’ (PN). PNs are basically composed of large aggregating chondroitin sulphate proteoglycans connected to a hyaluronan backbone, stabilized by link proteins and cross-linked via tenascin-R (TN-R). Under experimental conditions in mice, PN-ensheathed neurons are better protected against iron-induced neurodegeneration than neurons without PN. Still, it remains unclear whether these neuroprotective effects are directly mediated by the PNs or are associated with some other mechanism in these neurons unrelated to PNs. To identify molecular components that essentially mediate the neuroprotective aspect on PN-ensheathed neurons, we comparatively analysed neuronal degeneration induced by a single injection of FeCl3 on four different mice knockout strains, each being deficient for a different component of PNs. Aggrecan, link protein and TN-R were identified to be essential for the neuroprotective properties of PN, whereas the contribution of brevican was negligible. Our findings indicate that the protection of PN-ensheathed neurons is directly mediated by the net structure and that both the high negative charge and the correct interaction of net components are essential for their neuroprotective function.

No description available.

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Made available in DSpace on 2016-06-02T20:19:17Z (GMT). No. of bitstreams: 1 4141.pdf: 1483003 bytes, checksum: 97d4909d05c961c3bb73e05056aee7d3 (MD5) Previous issue date: 2012-02-29 / Financiadora de Estudos e Projetos / The aim of this study was to evaluate the effect of a strength exercise protocol on articular cartilage in animal model of osteoarthritis (OA). Thirty-six rats were divided in 6 groups: Control (C, n = 6), Osteoarthritis (OA, n = 6), Sham (S, n = 6), Control with exercise (E n = 6), Osteoarthritis with exercise (OAE, n = 6) and Sham with exercise (SE, n = 6). The animal model of osteoarthritis was anterior cruciate ligament transection (ACLT) in rats. After 2 weeks of ACLT, groups E, OAE and SE started the strength exercise protocol, three times a week for 8 weeks. We used the Mankin Histologic Grading System, measured the density of chondrocytes, the density of collagen fibers and the expression of collagen type II, chondroitin sulfate and fibronectin. The groups subjected to strength exercise protocol, OAE and SE, had lower values for the Mankin score, chondrocyte density and fibronectin expression and higher values for collagen fibers density, type II collagen expression and chondroitin sulfate expression when compared with OA and S, respectively. Group E was different from group C only in chondrocyte density and fibronectin expression. Therefore, strength exercise changes the content and expression of different articular cartilage constituent, having influence on our animal model of osteoarthritis and provides benefits to the cartilaginous tissue. / O objetivo desse estudo foi avaliar o efeito de um protocolo de exercício resistido na cartilagem articular de modelo animal de osteoartrite (OA). Trinta e seis ratos foram divididos em 6 grupos: Controle (C), Osteoartrite (OA); Sham (S), Exercício (E), Osteoartrite e Exercício (OAE), Sham e Exercício (SE). Os grupos OA, OAE, S e SE foram submetidos à transecção cirúrgica do ligamento cruzado anterior (LCA) do joelho esquerdo, sendo que somente os grupos OA e OAE tiveram o LCA seccionado. Após 2 semanas da cirurgia, os grupos E, OAE e SE iniciaram o protocolo de exercício resistido, 3 vezes por semana, durante 8 semanas. Foi aplicado o sistema de graduação histológica de Mankin, mensurado a densidade de condrócitos, a densidade de fibras colágenas e a expressão de colágeno tipo II, sulfato de condroitina e fibronectina. Os grupos submetidos ao protocolo de exercício resistido, OAE e SE, apresentaram menores valores para o sistema de graduação de Mankin, densidade de condrócitos e expressão de fibronectina e maiores valores para densidade de fibras colágenas, expressão de colágeno tipo II e sulfato de condroitina quando comparados aos grupos OA e S, respectivamente. O Grupo E apresentou diferença do grupo C somente na avaliação da densidade de condrócitos e na expressão de fibronectina. Portanto, o exercício resistido promove modificações no conteúdo e na expressão de diferentes constituintes da cartilagem articular, exercendo influência em nosso modelo de osteoartrite e trazendo benefícios para o tecido cartilaginoso.

Elastin-like peptides (ELPs) are stimulus-responsive protein-based polymers which are attractive material for biomedical research due to their biocompatibility and unique properties. The physical properties of ELPs are dependent on the chain length and the chosen amino acid at the guest residue position. This imparts unlimited flexibility in designing ELP based biomaterials with the desired physical properties. We have shown that in addition to their physical properties, ELPs have biological activities that are conducive to tissue regeneration. Specifically, we found that ELPs induce fibroblast proliferation via cell surface heparan sulfate proteoglycans (HSPG). Furthermore, our data suggests that ELP based materials with differential proliferative potential can be designed by controlling the interaction of ELPs with HSPGs by incorporating either hydrophobic or positively charged residues within the ELP sequence. Fibroblast proliferation is important for granulation tissue formation which is important in chronic wounds as well as in healing of other tissues. The customizable biological activity of ELPs coupled with their unique physical properties will enable us to design novel, sustainable and cost effective therapies for different tissue regeneration applications. ELPs can be genetically fused to biologically active peptides or proteins. These fusions can be expressed and readily purified since they maintain the phase transitioning property of the fused ELP domain. Moreover, depending on the ELP sequence chosen the chimeric fusion sequences can self-assemble into unique structures such as nanoparticles. These structures can then be applied to the injury site where they not only provide unique topographical cues or structural support but also act as delivery vehicles for the fused bioactive protein. We developed a multifunctional nanoparticle that is comprised of PMP-D2-ELP fusion protein and different functional peptide ELP fusion proteins to preserve the bioactivity of the functional group with the existence of elastase. These heterogeneous particles will be beneficial for the delivery of combination therapies to solve multiple problems that often existed in chronic wound healing or other tissue regeneration process. In summary, this study adds to our understanding of the biological activity of ELP and the interaction mechanism that allow the regulation of cellular behavior. Furthermore this work also investigated the potential therapeutic application of ELP as a delivery platform for chronic wound healing.