The primary objective of my thesis was to investigate the effect of crimp-like fibrous scaffolds on bovine fibroblasts and to develop a scaffold for anterior cruciate ligament (ACL) tissue engineering. To achieve this objective, fibrous biodegradable polymeric scaffolds were fabricated, which upon relaxation developed a crimp-like structure, which resembled the crimp seen in native collagen. The understanding of the crimp mechanism allowed for controlling crimp-like patterns in various polymer fibre systems, and was determined to be due to residual stress coupled with an operating temperature (Top) above the glass transition temperature of the polymer (Tg). The benefit of crimp was evaluated by seeding fibroblasts on crimp-like fibres that were subjected to dynamic mechanical loading. The results showed a significant increase in extracellular matrix (ECM) accumulation by fibroblasts that experienced crimp unfolding. In addition, fibroblasts seeded on mechanically stimulated crimp-like fibrous scaffolds formed ECM bundles that resembled collagen fibre fascicles. Two separate studies were conducted to fabricate fibrous scaffolds with high modulus: one on thermoplastic polyesters and the other on a photocrosslinkable polyester. Of the thermoplastic polyesters investigated, poly(L-lactide-co-D,L-lactide) P(LLA-DLLA) exhibited the highest modulus, and was the most resistant to hydrolytic degradation. These fibres were placed in a heated aqueous environment to exhibit a crimp-like pattern similar to that of native collagen. Bovine fibroblasts were shown to attach, proliferate and deposit ECM on the surface of the P(LLA-DLLA) fibrous scaffolds. In addition, the deposited ECM appeared to be organized in distinctive bundles that resembled fascicles found in native ACL. However, upon crimp unfolding the crimp was not completely recovered. Photocrosslinkable poly(L-lactide-co-trimethylene carbonate cinnamate) P(LLA-TMC cinnamate) fibres in addition to supporting cell proliferation and ECM accumulation, completely recovered their crimp-like pattern, via [2 + 2] cycloaddition of the cinnamate groups. The recovery of crimp upon unfolding is a novel design feature incorporated into electrospun fibres as it innately mimics the function of collagen fibres found in the ACL. From the results obtained it is evident that crimp and its unfolding are key design features/conditioning techniques that need to be incorporated into fibrous scaffolds that possess high modulus, intended for ligament tissue engineering. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2012-01-05 14:11:25.965

Osteoarthritis and osteochondral defects are ailments that are increasing in frequency as the lifespan of the population increases and sedentary lifestyle becomes more common. Osteoarthritis is an inflammatory disease that causes the progressive degeneration of articular surfaces and the underlying bone. Accidents and injuries can cause osteochondral defects similar to osteoarthritis. In both cases the structure of the articular cartilage fails, leading to pain and disability. Articular cartilage has a naturally poor ability to regenerate since there is no vasculature and it is aneural. The sparse chondrocytes mainly act to maintain the healthy extracellular matrix. Once the defect is severe enough, a surgical intervention becomes necessary. For small defects and young patients, a cell-based treatment can be used, whereas for larger defects and severe osteoarthritis a partial or whole joint arthroplasty is performed. Methods to repair osteochondral defects have been improving over the years as the inter-disciplinary understanding of joints, and what is required to repair them, has increased. However, there are still issues to solve in order to achieve consistently good results in both joint replacement and repair of cartilage. The main issue faced with current techniques used for joint replacement is poor integration of the artificial joint, leading to loosening at the bone interface over time, while cartilage repair techniques face the problem of generating mechanically inferior fibrocartilage. It is known that surface chemistry and structures at micro- and nanoscale influence cell behaviour, which can be utilised to guide their attachment, proliferation and phenotype. Scaffold-free approaches and mechanical stimulation have previously given promising results in generating articular neocartilage. This thesis aims at exploring tools and solutions to the problems involved in implant integration, chondrocyte expansion and neocartilage tissue engineering. We hypothesised that 1) ultra-short pulsed laser deposition can be used to create biocompatible coatings; 2) micropillars with nanoscale features can improve the maintenance of the chondrocyte phenotype in culture and 3) hypergravity can aid in the production of more native-like neocartilage constructs. Our studies showed that ultra-short pulsed laser ablation can be used to create various surfaces for studying cell behaviour. Cell viability was slightly higher on a rough titanium oxide, whereas the cell area was significantly smaller on rough titanium oxide, indicating a lower amount of focal adhesions. Nanopatterned microstructures were not capable of maintaining the chondrocyte phenotype in culture, but they were not disadvantageous either. Hypergravity might help in creating a native-like distribution of collagen and proteoglycans. The constructs were more uniform in shape, but biomechanically the constructs were not different from non-centrifuged controls.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is one of the most common sites for breast cancer metastasis. Osteocytes compose approximately 90% of the cell population in bone matrix. Osteocytes are very sensitive to mechanical stimulation, and physical activities play an essential role in maintaining bone's health. Mechanical stimulation can alter the gene expression profile in osteocytes. However, little is known about the effects of mechanical stimulation on tumor-bone interactions. In this thesis, this question has been addressed: Does applying mechanical stimulation to osteocytes change tumor-osteocytes interactions? The hypothesis is that mechanical stimulation can change osteocytes secreting signals and contribute to higher proliferation and migration of tumor cells. In this thesis, fluid flow-driven shear stress has been used as the mechanical stimulator for osteocytes, and the interactions of tumor-osteocytes, with and without mechanical stimulation has been investigated. Monolayer cultures and 3D spheroids of breast cancer cells, including TMD and 4T1 cells were cultured in the conditioned medium (CM) isolated from MLO-A5 osteocytes, and fluid flow-treated conditioned medium (FFCM), and their migratory behavior, proliferation, and protein expression have been evaluated. The results showed that in response to MLO-A5 FFCM, tumor cells behave differently in Src expression, proliferation, and migration compared to MLO-A5 CM. As opposed to MLO-A5 CM, FFCM promoted migration, reduced proliferation, and upregulated Src expression in tumor cells. Moreover, by plasmid and siRNA transfection it has been shown that Src is upstream of Snail and their upregulation is causing epithelial-mesenchymal transition(EMT) responses in tumor cells. Furthermore, ELISA concentration assessment showed the involvement of TGF-beta in Src upregulation. An in vivo study using seventeen mice was conducted to investigate the effect of mechanical stimulation on clinical conditions. Compressive loads were applied to tibia after intratibial injection of 4T1.2 cells. The results suggested that direct mechanical stimulation of metastasized bone, might not be advantageous, and cause more damage. Furthermore, the results indicated that direct mechanical loading can make the knee joint more fragile. This research showed mechanical stimulation can cause tumor cells to behave more migratory in bone microenvironment, and demonstrated its crucial role in tumor-osteocytes interactions.

Le cartilage articulaire est composé de chondrocytes et d'une matrice extracellulaire organisés de manière stratifiée dans l'épaisseur du tissu. Ce tissu ne se régénère pas de manière efficace après une lésion. L'objectif de ce travail est de construire par pulvérisation des hydrogels à base d'alginate et de film multicouches de polyélectrolytes pour créer in vitro un néotissu pouvant combler des lésions de cartilage articulaire. La méthode a été validée en observant une bonne viabilité et une synthèse matricielle par les cellules, et de meilleures propriétés mécaniques des hydrogels pulvérisés à 0,9 bar par rapport au moulage. Après la pulvérisation de cellules souches mésenchymateuses, les résultats ont montré une bonne viabilité et une différenciation des cellules. Puis, des hydrogels bistratifiés ont été construits et cultivés jusqu'à 56 jours sans dissociation des couches et sans migration des cellules. Enfin, les hydrogels ont été fonctionnalisés en modifiant la composition des couches et en y appliquant des stimulations mécaniques. Les propriétés mécaniques des hydrogels varient en fonction de leur composition et sont meilleures pour ceux stratifiés. De plus, leur stimulation mécanique a permis de potentialiser l'effet du biomatériau sur la différenciation des cellules. En conclusion, cette étude montre que des cellules souches mésenchymateuses ensemencées dans un hydrogel bistratifié pulvérisé sont fonctionnelles en termes de différenciation chondrocytaire et de synthèse matricielle. Les propriétés mécaniques des hydrogels stratifiés ne sont pas altérées. De plus, la stimulation mécanique a potentialisé la différenciation des cellules / The articular cartilage is composed of chondrocytes and of a specific extracellular matrix which are organized depth-dependently. The tissue did not have an efficient self-renewal of defects. The purpose of this study is to build up layer-by-layer a stratified hydrogel by alternating gels and multilayers polyelectrolytes film spraying, in order to obtain a neotissu in vitro to fill lesions. First, the process was validated by observing a good cells viability and matrix synthesis, and stronger mechanical behaviors of sprayed hydrogels compared to molded one. Secondly, after their spraying, mesenchymal stem cells still have a good viability and their differentiation potential. Then, bistratified scaffolds were built up and cultured up to 56 days without layers dissociation and without cells migration between layers. Finally, scaffolds were functionalized by changing biomaterial composition and by applying mechanicals stimulations. Results show us not only that the composition influences the mechanical behavior of the hydrogel, but that the stratification did not affect it. Furthermore, mechanicals stimulations improve stem cells differentiation in function of biomaterials compositions. In conclusion, this study proves not only that we are able to build up stratified scaffold seeded with mesenchymal stem cells which still have their differentiation capability and synthesize matrix, but that mechanical behaviors are improved after the biomaterial spraying and not alter by the stratification. Moreover, mechanical stimulation applied to the scaffold improves the differentiation of mesenchymal stem cells to a chondrogenic phenotype

Contexte – La stimulation de la muqueuse trachéale peut provoquer une réponse défensive à type de toux. La finalité de cette réponse est la protection des voies aériennes via la clairance du mucus et des particules déposées au sein de l’arbre trachéo-bronchique. La stimulation de la muqueuse nasale est à l’origine de réponses défensives n’incluant toutefois pas la toux. La modulation de la toux suggère de possibles interactions centrales des afférences provenant de localisations anatomiques distinctes. Objectif – Déterminer si une stimulation trachéale mécanique discriminante est capable de provoquer une toux lors d’une apnée provoquée par l’instillation nasale d’eau distillée. Méthodes – Douze lapins anesthésiés et trachéotomisés ont été étudiés. Les stimulations trachéales mécaniques ont été réalisées dans 3 conditions : contrôle, après instillation nasale de sérum physiologique, et lors d’apnée suite à l’instillation d’eau distillée. Résultats – Les paramètres ventilatoires de références ne sont pas différents dans les 3 conditions. Un total de 171 stimulations trachéales a été réalisé. Lors de l’apnée, 81% des réponses sont des réflexes expiratoires et le pic de débit expiré est inférieur (p < 0.0001) à celui observé dans les conditions contrôle et sérum physiologique. L’incidence des réponses comprenant un réflexe de toux est plus faible (p < 0,0001) en cas d’instillation d’eau distillée que dans les 2 autres conditions.Conclusion – La stimulation nasale par l’eau distillée désensibilise les réflexes de défense respiratoire faisant suite à une stimulation trachéale mécanique / Context - Cough may be triggered by irritation of afferents located in the airway mucosa. Primary role is to expel inhaled foreign matter from the lungs or clear the airways of endogenous mucus. Stimulation of the nasal mucosa provokes defensive responses but not cough. The ‘cough center’ can be tuned by various afferent inputs, suggesting possible interactions at a central level of neural pathways originating from distant anatomical sites. Objective - The present study was designed to determine whether brief mechanical stimulation of the trachea could trigger cough during apnoea elicited by nasal instillation of water. Method - Twelve anesthetized, tracheotomized rabbits were studied. Mechanical stimulation of the trachea was performed under 3 conditions: baseline control, after instillation of saline into the nose and during apnoea following instillation of water. Results - Baseline breathing pattern did not differ between the 3 conditions. In a series of 171 stimulations, expiration reflex occurred in 81% of stimulations during apnoea with a significantly (p < 0.0001) lower peak expiratory flow than at baseline or during saline instillation. The incidence of responses comprising a cough reflex was also lower during water instillation than at either baseline or with saline (p < 0.0001). Conclusion - These results indicate that stimulation of nasal afferents with distilled water likely down-regulates cough

Disease and degeneration of articular cartilage and fibrocartilage tissues severely compromise the quality of life for millions of people. Although current surgical repair techniques can address symptoms in the short term, they do not adequately treat degenerative joint diseases such as osteoarthritis. Thus, novel tissue engineering strategies may be necessary to combat disease progression and repair or replace damaged tissue. Both articular cartilage and the meniscal fibrocartilage in the knee joint are subjected to a complex mechanical environment consisting of compressive, shear, and tensile forces. Therefore, engineered replacement tissues must be both mechanically and biologically competent to function after implantation. The goal of this work was to investigate the effects of oscillatory tensile loading on three dimensional engineered cartilaginous tissues in an effort to elucidate important aspects of chondrocyte and fibrochondrocyte mechanobiology. To investigate the metabolic responses of articular chondrocytes and meniscal fibrochondrocytes to oscillatory tensile loading, various protocols were used to identify stimulatory parameters. Several days of continuously applied tensile loading inhibited extracellular matrix metabolism, whereas short durations and intermittently applied loading could stimulate matrix production. Subpopulations of chondrocytes, separated based on their zonal origin within the tissue, differentially responded to tensile loading. Proteoglycan synthesis was enhanced in superficial zone cells, but the molecular structure of these molecules was not affected. In contrast, neither total proteoglycan nor protein synthesis levels of middle and deep zone chondrocytes were substantially affected by tensile loading; however, the sizes of these new matrix molecules were altered. Up to 14 days of intermittently applied oscillatory tensile loading induced modest increases in construct mechanical properties, but longer durations adversely affected these mechanical properties and increased degradative enzyme activity. These results provide insights into cartilage and fibrocartilage mechanobiology by elucidating cellular responses to tensile mechanical stimulation, which previously had not been widely explored for these tissues. Understanding the role that mechanical stimuli such as tension can play in the generation of engineered cartilaginous tissues will further the goal of developing successful treatment strategies for degenerative joint diseases.

The current study characterizes the mechanical and biodegradation properties of a polycarbonate polyurethane (PU) electrospun nanofiber scaffold intended for use in the growth of a tissue engineered annulus fibrosus (AF) intervertebral disc component. Both the tensile strength and initial modulus of aligned scaffolds were higher than those of random scaffolds and remained unaffected during a 4 week biodegradation study, suggesting a surface-mediated degradation mechanism. The resulting degradation products were non-toxic. Confined compressive mechanical force of 1kPa, was applied at 1Hz to in vitro bovine AF tissue grown on the scaffolds to investigate the influence of mechanical force on AF tissue production, which was found to decrease significantly at 72 hours relative to 24 hours, independent of any effects from mechanical forces. Overall, the consistent rate of PU degradation, along with mechanical properties comparable to those of native AF tissue, and the absence of cytotoxic effects, make this polymer suitable for further investigation for use in tissue-engineering the AF.

The current study characterizes the mechanical and biodegradation properties of a polycarbonate polyurethane (PU) electrospun nanofiber scaffold intended for use in the growth of a tissue engineered annulus fibrosus (AF) intervertebral disc component. Both the tensile strength and initial modulus of aligned scaffolds were higher than those of random scaffolds and remained unaffected during a 4 week biodegradation study, suggesting a surface-mediated degradation mechanism. The resulting degradation products were non-toxic. Confined compressive mechanical force of 1kPa, was applied at 1Hz to in vitro bovine AF tissue grown on the scaffolds to investigate the influence of mechanical force on AF tissue production, which was found to decrease significantly at 72 hours relative to 24 hours, independent of any effects from mechanical forces. Overall, the consistent rate of PU degradation, along with mechanical properties comparable to those of native AF tissue, and the absence of cytotoxic effects, make this polymer suitable for further investigation for use in tissue-engineering the AF.

No description available.