Extrinsic repair techniques for the treatment of acute chondral injuries continue to yield suboptimal repair. The inability of these techniques to produce hyaline cartilage underscores the limitations in our understanding of basic chondrocyte biology. Conversely, intrinsic repair tissue has not been extensively studied despite the fact that it can yield hyaline-like cartilage and is commonly observed in osteoarthritis. Attempts at extrinsic repair could therefore benefit from a better understanding of the successes and failures inherent in the intrinsic repair process. Chondrocyte culture has typically been conducted under non-physiologic conditions whereby chondrocytes readily dedifferentiate. Consequently, much of the knowledge gained about chondrocytes has been misleading thus hindering advancements in chondrocyte biology and attempts at extrinsic articular cartilage (AC) repair. Hypoxic culture conditions, which are beneficial towards the preservation of the chondrocyte phenotype, remain insufficient due to elevated collagen I gene expression. As such, an appropriate model system does not yet exist in which to study physiologically-relevant chondrocyte biology. The presence and prevalence of collagen I in both degenerate and de novo osteoartritic tissue was examined immunohistochemically. Collagen I deposition during osteoarthritic progression was compared against IHC staining for collagen II and aggrecan. A novel model system was also evaluated for chondrocytic phenotype retention. To this end, hypoxic, high-density-monolayer-chondrocyte (HDMC) cultures were compared to freshly isolated chondrocytes for their ability to maintain a chondrocytic extracellular matrix (ECM) gene expression profile. HDMC culture conditions prevented the severe loss of the phenotype typically associated with conventional monolayer culture. Moreover, prolonged HDMC culture resulted in the formation of a complex ECM and a marked suppression of collagen I expression. This study also demonstrated that collagen I deposition occurs in osteoarthritic AC at the onset of structural damage and increases in response to increasing structural damage. Collagen I deposition was also found in different types of de novo cartilage associated with osteoarthritic joints and suggests that it plays an important role in intrinsic cartilage repair. Taken together, this work demonstrates that collagen I is a common feature in the ECM of structurally immature and structurally damaged AC and hence may play a role in tissue stabilization. / Experimental Surgery

No description available.

The inability to accurately reproduce the complexities of the in vivo tumor microenvironment with reductionist-based two-dimensional in vitro cell culture models has been a notable deterrent in identifying therapeutic agents that reliably translate to in vivo animal and human clinical trials. In an effort to address this, a growing number of three-dimensional (3D) in vitro tumor models capable of mimicking specific tumorigenic processes have emerged within the last decade. This concept stems from the understanding that cells cultured within 3D in vitro matrices have the ability to acquire phenotypes representative of the in vivo microenvironment. The objective of this project was to apply a tissue engineering approach towards developing a 3D in vitro tumor angiogenesis model. Initially, different scaffolds were investigated for supporting 3D tumor growth, including bacterial cellulose, electrospun polycaprolactone/collagen I, and highly porous electrospun poly(L-lactic acid). However, cancer cells cultured on these scaffolds demonstrated poor adhesion, sufficient adhesion with poor infiltration, and increased but still inadequate infiltration, respectively. Collagen I hydrogels were chosen as an appropriate scaffold for facilitating 3D in vitro tumor growth for two reasons -- cell-mediated degradation and immediate 3D cell growth. It was hypothesized that cancer cells cultured within collagen I hydrogels could be encouraged to recapitulate key characteristics of in vivo tumor progression. MDA-MB-231 human breast cancer cells were shown to experience hypoxia and undergo necrosis in response to limitations in oxygen diffusion and competition for nutrients. Upregulation of hypoxia-inducible factor-1" resulted in a significant increase in vascular endothelial growth factor gene expression. To capitalize on this endogenous angiogenic potential, microvascular endothelial cells were cultured on the surface of the designated "bioengineered tumors." It was hypothesized that paracrine signaling between tumor and endothelial cells co-cultured within this system would be sufficient for inducing an angiogenic response in the absence of exogenous pro-angiogenic growth factors. Endothelial cells in the co-culture group were shown to invasively sprout into the underlying collagen matrix, forming a capillary-like tubule network. This project culminated with the establishment of an improved in vitro tumor model that can be used as a tool for accurate evaluation and refinement of cancer therapies. / Ph. D.

In der vorliegenden Arbeit wurde der Einfluss der humanen AT2-Rezeptorexpression und -stimulation auf den Collagen I alpha 2-mRNA-Gehalt und die Migration von porcinen kardialen Fibroblasten untersucht, um die Frage zu klären, ob AT2-Rezeptoren in kultivierten kardialen Fibroblasten AT1-antagonistische antifibrotische und migrationshemmende Effekte auf den Collagen I alpha 2-mRNA-Gehalt bzw. die Migration ausüben. Um die Funktion der AT2-Rezeptoren in der Zellkultur untersuchen zu können, wurde die AT2-cDNA durch adenovirale Transduktion in die Fibroblasten übertragen und so der AT2-Rezeptor überexprimiert. Mittels RT-PCR wurden die relativen Änderungen im Collagen I alpha 2-mRNA-Gehalt in TGF-beta1- bzw. TGF-beta1 plus Ang II-stimulierten Fibroblasten im Vergleich zur unstimulierten Kontrolle bestimmt. Alle Werte wurden auf ein Referenzgen (beta-Actin) bezogen. Die AT2-Stimulation änderte den relativen Collagen I alpha 2-mRNA-Gehalt der Fibroblasten nicht signifikant gegenüber den Antisense-(Ad5TA2)-transduzierten Fibroblasten. In der modifizierten Boyden-Kammer wurde der AT2-vermittelte Effekt von Ang II, hPDGF-BB sowie der Kombination beider Stoffe auf die Migration untersucht. Die alleinige Stimulation von AT2-Rezeptoren mit Ang II verhinderte die Migration gegenüber nichttransduzierten Fibroblasten. In Kombination mit hPDGF-BB änderte Ang II die Migration in AT2-überexprimierenden Fibroblasten nicht gegenüber den Antisense-(Ad5TA2)-transduzierten Fibroblasten. Bei ausschließlicher Stimulation durch hPDGF-BB wurde aber in AT2-exprimierenden Fibroblasten eine signifikant geringere Migration als in Antisense-(Ad5TA2)-transduzierten Fibroblasten festgestellt. Die zugrunde liegende Hypothese, dass AT2-Expression und Stimulation den relativen Collagen I alpha 2-mRNA-Gehalt hemmt, konnte in den vorliegenden Experimenten nicht bestätigt werden. Dies ließ keine inhibitorische AT2-vermittelte Wirkung von Ang II im Bezug auf den TGF-beta1-induzierten Collagen I alpha 2-mRNA-Gehalt erkennen. Dagegen führte die Ang II-Stimulation überexprimierter AT2-Rezeptoren zu einer verringerten Migration und vermittelte so einen AT1-antagonistischen Effekt. / In this work the influence of expression and stimulation of the human AT2 receptor on Collagen I alpha 2-mRNA-content and migration of porcine cardiac fibroblastst was tested to clarify the question if AT2 receptors promote AT1 antagonistic antifibrotic and antimigratory effects on collagen I alpha 2-mRNA content and migration. To examine the AT2 receptor function in the cell culture AT2 cDNA was transferred into fibroblasts by adenoviral transduction and the AT2 receptor was overexpressed. Through the use of RT-PCR the relative changes in collagen I alpha 2-mRNA content in TGF-beta1 stimulated and TGF-beta1 plus Ang II stimulated fibroblasts were assayed and compared to the unstimulated control. All values were referred to a reference gene (beta-actin). Stimulation of AT2 receptors did not change the relative collagen I alpha 2-mRNA content of the fibroblasts significantly compared to antisense-(Ad5TA2) transduced fibroblasts. In the modified Boyden-chamber the AT2 mediated effect of Ang II, hPDGF-BB and the combination of both on migration was assessed. The stimulation of AT2 receptors with Ang II inhibited migration compared to nontransduced fibroblasts. In combination with hPDGF-BB Ang II did not change the migration in AT2 overexpressing fibroblasts compared to antisense-(Ad5TA2)-transduced fibroblasts. In the case of exclusive stimulation of AT2-expressing fibroblasts with hPDGF-BB a significantly lower migration was found compared to antisense-(Ad5TA2)-transduced fibroblasts. The underlying therory that AT2 expression and stimulation inhibits the relative collagen I alpha 2-mRNA content could not be confirmed in this work. This did not reveal an inhibitory AT2 mediated effect of Ang II in respect to the TGF-beta1 induced collagen I alpha 2-mRNA content. In contrast to that Ang II stimulation of overexpressed AT2 receptors led to a decreased migration and mediated an AT1 antagonistic effect.

L’objectif de cette thèse a consisté à étudier in vitro des phénomènes d’auto-assemblage de molécules de collagène de type I natif, dénaturé ou modifié. Dans une première partie, nous avons analysé des solutions denses en milieu acide de collagène I et de gélatine A par microscopie à lumière polarisée et microscopie SHG résolue en polarisation (P-SHG). Nous avons ainsi mis en évidence la première mésophase obtenue à partir de gélatine. Nous avons ensuite étudié l’impact de la gélatine sur des mésophases en contre-plaqué de collagène I et montré que le collagène pouvait être subtilisé par la gélatine à hauteur de 20% sans en affecter la formation des phases en contre-plaqué. Les matrices collagène/gélatine obtenues après fibrillogenèse ont été caractérisées à différentes échelles en ayant recours à la calorimétrie différentielle (DSC), à la microscopie électronique à transmission (MET), au P-SHG et l’imagerie par résonnance magnétique (IRM). Nous avons montré que ces matrices présentent une structuration hiérarchique de type fractal et que la gélatine stabilise le collagène.Dans la seconde partie, nous nous sommes intéressés aux propriétés d’auto-assemblage de molécules de collagène I modifié avec de la rifamycine SV pour la synthèse de gels à délivrance contrôlée. Nous avons utilisé différents modes d’assemblage afin d’arriver à notre but et avons caractériser les propriétés chimiques, rhéologiques et antibiotiques des gels obtenues. Pour mieux comprendre le système, nous avons étudié la structure des assemblages de collagène modifié au sein d’un système dilué à l’aide du cryo-MET et de la microscopie à force atomique couplé à la spectroscopie infrarouge (AFMIR). / The object of this thesis was to study the in vitro self-assembly phenomena of native, denatured or modified collagen type I.In the first part, we analysed dense solutions of collagen I and gelatin A (in acidic medium) by polarized light microscopy and polarization resolved SHG microscopy (P-SHG). We have thus observed the first mesophase obtained from gelatin. We then studied the impact of gelatin on collagen I plywood mesophases and showed that collagen could be replaced by gelatin up to 20% without affecting the formation of the plywood phases.Collagen/gelatin matrices obtained after fibrillogenesis were characterized at different scales by using Differential Scanning Calorimetry (DSC), Transmission Electron Microscopy (TEM), P-SHG and Magnetic Resonance Imaging (MRI). We showed that these matrices have a hierarchical structure of fractal type and that gelatin stabilizes collagen.In the second part, we studied the self-assembly of collagen I modified with rifamycin SV for the synthesis of controlled delivery gels.We used different methods of assembly in order to reach our goal and we characterized the chemical, rheological and antibiotic properties of the obtained gels. To better understand the system, we studied the structure of modified collagen assemblies in a diluted systems with cryo-TEM and atomic force microscopy coupled with infrared spectroscopy (AFMIR).

Three-dimensional fibrillar networks reconstituted from collagen I are widely used as biomimetic scaffolds for in vitro and in vivo cell studies. Various physicochemical parameters of buffer conditions for in vitro fibril formation are well known, including pH-value, ion concentrations and temperature. However, there is a lack of a detailed understanding of reconstituting well-defined 3D network topologies, which is required to mimic specific properties of the native extracellular matrix. We screened a wide range of relevant physicochemical buffer conditions and characterized the topology of the reconstituted 3D networks in terms of mean pore size and fibril diameter. A congruent analysis of fibril formation kinetics by turbidimetry revealed the adjustment of the lateral growth phase of fibrils by buffer conditions to be key in the determination of pore size and fibril diameter of the networks. Although the kinetics of nucleation and linear growth phase were affected by buffer conditions as well, network topology was independent of those two growth phases. Overall, the results of our study provide necessary insights into how to engineer 3D collagen matrices with an independent control over topology parameters, in order to mimic in vivo tissues in in vitro experiments and tissue engineering applications.

No description available.

Every year there are nearly two million new cases of invasive breast cancer worldwide and over 500,000 deaths, the majority from metastatic sites. While cellular changes during tumorigenesis and progression have been studied, our understanding of extracellular matrix remodeling, at the fiber level, by individual and collective cellular cohorts remains limited. Furthermore, recent studies suggest that there is a correlation between the organization of collagen I fibers perpendicular to the tumor and patient survival. However, the underlying mechanism of this alignment remains unknown. The central hypothesis proposed in this dissertation is that breast cancer tumors reorganize collagen I fibers perpendicular to the tumor surface via integrin β1 and matrix metalloproteinases (MMPs). To investigate this hypothesis, we developed a novel in vitro assay that replicates collagen I fiber alignment previously reported in vivo and a new quantitative collagen I fiber orientation algorithm. Our studies using multicellular aggregates, derived from the triple negative breast cancer cell line MDA-MB-231, embedded into collagen I matrices and confocal reflectance microscopy provide novel insights into how the local microenvironment is affected and into local orientation of the collagen I fibers near the spheroid-collagen I interface. These results agree well with our computational studies. Furthermore, the viability of the algorithm is demonstrated using both in silico and in vitro derived images, and shows that this algorithm is more accurate than similar algorithms previously published. Using the developed in vitro assay and computational algorithm it is also demonstrated that knocking down integrin β1 reduces the amount of collagen I aligned perpendicularly to the tumor surface, while inhibiting MMP activity using the broad spectrum MMP inhibitor GM6001 increases the amount of collagen I aligned perpendicularly to the tumor surface at early time points. The work presented here has implications in three-dimensional multicellular assays, accurate fiber orientation analysis, and understanding the role of integrins in matrix reorganization and cancer cell metastasis. / 2019-08-09T00:00:00Z

Understanding how insulin-like growth factor-I (IGF-I) signaling in mammary epithelial cells may be modified or interrupted by modifications in the cellular environment may lead to 1) methods to increase the growth and proliferation of normal mammary epithelial cells for an increase in the amount of milk produced on a per animal basis or to 2) the development of medical interventions to disrupt the growth and proliferation of cancerous mammary epithelial cells. IGF-I, a signaling protein provided by stromal cells and through the bloodstream, stimulates the proliferation of mammary epithelial cells and is crucial for mammary development. Collagen I is an extracellular matrix protein (ECM) found in skin and in other connective tissues throughout the body. The guiding question in this dissertation was how IGF-I signaling and how binding protein profile were influenced by autocrine IGF-I and by collagen I. The MAC-T cell line was chosen as the cell model utilized in these investigations because it is an immortalized bovine mammary epithelial cell line known to retain hormonal responsiveness to IGF-I. It was hypothesized that the production of IGF-I by mammary epithelial cells (autocrine secretion) would alter the response of these cells to additional IGF-I by de-sensitizing the IGF-I receptor on the cell surface. The normal mammary epithelial cell does not produce IGF-I and responds to IGF-I supplied either by stromal cells (paracrine pathway) or through the bloodstream (endocrine pathway). The IGF-I secreting bovine mammary epithelial cell line was investigated for the response of the cells to autocrine IGF-I, and the response was compared to the normal, parental cell line. To examine the effect of autocrine IGF-I on the cells, IGF-I was added both to MAC-T cells and to cells transfected to secrete IGF-I (SV40-IGF-I). The cell response of the two cell lines was compared using microphysiometry, a tool that measures IGF-IR stimulation by detecting resultant extracellular acidification. It was found that the SV40-IGF-I cell line retains IGF-I receptor sensitivity, yet, unlike the parental cell line, does not proliferate in response to IGF-I. Both cell lines exhibited increased protein synthesis in response to IGF-I as measured by amino acid uptake (AIB incorporation), but the lack of a proliferation response to additional IGF-I in the SV40-IGF-I cell line suggested that the autocrine cell line exhibited an un-coupling of IGF-IR stimulation with downstream cell proliferation. Both autocrine IGF-I and added IGF-I increased the amount of IGFBP-3 secreted by the cells into growth media. Additionally, it was hypothesized that the presence of collagen I, an important ECM protein, would alter the cell production of insulin-like binding protein-3 (IGFBP-3), a protein that modulates IGF-I interaction with the IGF-I receptor (IGF-IR). The literature reports that surface substrate can affect the phenotypic expression of cells, presumably via interaction with integrins, the cell surface receptors that connect cells to ECM proteins and that are responsible for cell adhesion and for cell migration. It was hypothesized that the MAC-T cells would interact with a collagen I surface (possibly via the a2b1 integrin) and that the stimulation of this transmembrane signaling molecule would in turn impact the IGF-I signaling pathway. Comparison studies on tissue culture plastic, collagen I BIOCOAT, and collagen I gel were performed. It was found that collagen I gel increased IGFBP-3 secretion and decreased insulin-like binding protein-2 (IGFBP-2) secretion in MAC-T cells. The collagen I BIOCOAT did not induce this response. Additional studies were performed to determine if there were differences in IGF-IR phosphorylation, exogenous IGF-I utilization, and IGFBP mRNA production by cells cultured on the three different substrates. IGF-IR phosphorylation was only evident following the addition of IGF-I to MAC-T cells on all three substrates. Measurement of residual IGF-I present in the cultured media of cells on all three substrates by radioimmunoassay did not reveal any differences in the amount of IGF-I present. Northern blot analysis revealed that the addition of IGF-I caused an increase in detected IGFBP-3 mRNA and a decrease in detected IGFBP-2 mRNA across all three surfaces. As measured by ligand blot analysis, cells cultured on all three surfaces showed an increase in IGFBP-3 protein in the media with IGF-I addition, and the collagen I gel showed more IGFBP-3 protein than the other two surfaces. However, cells cultured on collagen I gel showed a decrease in IGFBP-2 protein expression compared to cells cultured on tissue culture both with and without the addition of IGF-I. Cells cultured on tissue culture plastic and on collagen I BIOCOAT did not show a decrease in IGFBP-2 to correspond with the decreased IGFBP-2 mRNA detected in the presence of IGF-I on all three substrates. DNA assays to detect cell proliferation revealed no differences in cell DNA content in the absence of exogenous IGF-I and revealed similar increases in response to IGF-I addition on all three substrates. In conclusion, it was found that autocrine IGF-I un-couples increased IGF-IR stimulation by exogenous IGF-I from a downstream cell proliferation response. IGFBP-3 inhibits the ability of IGF-I to interact with the IGF-IR in MAC-T cells and inhibits subsequent cell proliferation. Collagen I gel increases IGFBP-3 secretion and decreases IGFBP-2 secretion by MAC-T cells. The relevance of this work is that it adds to the body of knowledge in understanding cellular function in mammary epithelial cells. It is known that the growth and the maintenance of living tissue are dependent on an intricate system of intercellular and intracellular responses which are orchestrated by the movement and secretion of proteins and other molecules. Goals of understanding mammary epithelial cell function include having the means to find ways to increase cell functionality via bioengineering and having the means to find ways to restore cells to normal function in disease processes such as cancer. / Ph. D.

<p>Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy in man with stromal fibrosis as one of the main features. Carcinoma cells synthesized no or little collagen I protein. Pro-α1(I) collagen mRNA was expressed by stromal cells throughout the tumor, but expression of procollagen type I protein was restricted to stromal cells situated close to nests of carcinoma cells. These data suggest that the carcinoma cells stimulated collagen type I deposition by increasing pro-α1(1) collagen mRNA translation. </p><p>Cocultures, of the human ATC cell line KAT-4, with fibroblasts under conditions that allow the study of stimulatory factors on collagen mRNA translation, showed that the KAT-4 cells stimulated collagen type I protein synthesis in fibroblasts. Specific inhibitors of PDGF and TGF-β1 and -β3 were able to inhibit this carcinoma cell-induced stimulation of collagen type I synthesis. These findings suggest that tumor cells were able to stimulate collagen mRNA translation in stromal fibroblasts by, at least in part, transferring PDGF and/or TGF-β1 and -β3.</p><p>Xenograft transplantation of different ATC cell lines into athymic mice demonstrated that the low collagen producing carcinoma cell lines were less tumorigenic compared to non-collagen producing carcinoma cell lines. The morphology of tumors derived from non-collagen producing ATC cell lines showed a well demarked stroma surrounding carcinoma cell nests. </p><p>TGF-β1 and -β3 were found to play a role in generating a high tumor interstitial fluid pressure (TIPF) in experimental KAT-4 tumors. A specific inhibitor of TGF-β1 and -β3 was able to lower TIPF and reduce tumor growth after a prolonged period of treatment, suggesting that TGF-β1 and -β3 have a role in maintaining a stroma that support tumor growth.</p>