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Additive manufacture of tissue engineering scaffolds for bone and cartilageEshraghi, Shaun 07 January 2016 (has links)
Bone and cartilage constructs are often plagued with mechanical failure, poor nutrient transport, poor tissue ingrowth, and necrosis of embedded cells. However, advances in computer aided design (CAD) and computational modeling enable the design of scaffolds with complex internal michroarchitectures and the a priori prediction of their transport and mechanical properties, such that the design of constructs satisfying the needs of the tissue environment can be optimized. The goal of this research is to investigate the capability of additive manufacturing technologies to create designed microarchitectured tissue engineering scaffolds for bone and cartilage regeneration. This goal will be achieved by pursuing the following two objectives: (1) the manufacture of bioresorbable thermoplastic scaffolds by selective laser sintering (SLS) (2) and the manufacture of hydrogel scaffolds by large area maskless photopolymerization (LAMP). SLS is a laser based additive manufacturing method in which an object is built layer-by-layer by fusing powdered material using a computer-controlled scanning laser. LAMP is a massively parallel ultraviolet curing-based process that can be used to create hydrogels from a photomonomer on a large-scale (558x558mm) while maintaining extremely high feature resolution (20µm). In this research, SLS is used to process polycaprolactone (PCL) and composites of PCL with hydroxyapatite (HA) for bone tissue engineering applications while LAMP is used to process polyethylene glycol diacrylate (PEGDA) which can be used for hard and soft tissue applications.
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Ionenstrahluntersuchungen am GelenkknorpelReinert, Tilo 17 February 2016 (has links) (PDF)
Knorpel ist ein kompliziertes System aus einem kollagenen Netzwerk, gefüllt mit wasserbindenden Makromolekülen (Proteoglykanen) und darin eingebetteten Zellen. Störungen in den komplexen Wechselbeziehungen können zur Gefährdung der strukturellen Integrität des Knorpels führen. Die hochauflösende Magnetresonanztomographie (NMR-Mikroskopie) kann über die Analyse der Signalintensität interne Knorpelstrukturen darstellen (hypo- und hyperintense Zonen).
Mit Hilfe ionenmikroskopischer Analysemethoden (PIXE, RBS, ERDA) wurden im Knorpel (femorale und tibiale Kondyle des Hausschweins) im Querschnitt die zweidimensionalen Verteilungen der Knorpelelemente (H, C, N, O, P, S, Cl, K und Ca) aufgenommen sowie die Konzentrationen in ausgewählten Zonen bestimmt. Ergänzend wurde mit STIM die Dichteverteilung im Knorpel untersucht. Es gelang auch mit STIM, erstmalig kollagene Fasern in ihrer, bis auf den Wasserentzug natürlichen, Umgebung im Knorpel und damit unverändert in ihrer Anordnung sichtbar zu machen (keine chemische Demaskierung nötig).
Die Ergebnisse wurden mit NMR- und polarisationsmikroskopischen Untersuchungen verglichen und in ihrem Zusammenhang mit den histologischen Knorpelzonen diskutiert. In den NMR-hypointensen Zonen fanden sich eine erhöhte Chlorkonzentration und punktförmige Calciumanreicherungen. Diese Zonen waren (im gefriergetrockneten Zustand) durch eine, bis zu einem Faktor vier höhere Dichte gekennzeichnet, die im maximalen Gehalt der Matrixelemente, H, C, N, O, (höchste Kollagendichte) begründet liegt. Im tibialen Knorpel konnten in der NMR-hypointensen Zone radial verlaufende einzelne Kollagenfasern nachgewiesen werden. Im femoralen Knorpel wurden in dieser Zone keine Einzelfasern nachgewiesen. Es deutete sich eine tubuläre Anordnung der Kollagenfasern an. In der hypertrophen Zone zeigten sich hohe Konzentrationen an Phosphor (Zellorganellen), Schwefel (Proteoglykane), Kalium (alkalisches Milieu) und Calcium (Vorstufe der Kalzifizierung). Die Chlorkonzentration hatte dort ihr Minimum. In dieser Zone verlaufen die Kollagenfasern radial und münden senkrecht in den Kalkknorpel. In der Tangentialfaserschicht wurde eine erhöhte Konzentration an Calcium und Phosphor beobachtet (Einlagerung von Calciumphosphaten). In dieser Zone wurden tangential verlaufende Kollagenfasern und ihr Übergang zur stärkeren Vernetzung mit teilweise arkadenförmiger Überstruktur sichtbar gemacht.
Zur genaueren Aufklärung der dreidimensionalen Anordnung der Kollagenen Strukturen wurden erste Experimente zur STIM-Tomographie durchgeführt.
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Dating death : forensic taphonomy and the postmortem intervalRogers, Christopher January 2010 (has links)
Determining the postmortem interval (PMI) remains one of the most important but challenging factors to establish in a suspicious death investigation. Unfortunately, as time passes current methods lose accuracy and only allow investigators to approximate how long ago death occurred. Bodies interred in clandestine graves prove particularly challenging due to an abundance of variables that need to be taken into consideration. Due to the problems associated with determining the PMI of buried remains this study will utilise macroscopic, microscopic, molecular, chemical and microbiological analyses to systematically document the decompositional changes to human hair and porcine cartilage and bone in a burial environment. The aim was to correlate decompositional changes with time and develop new methods for estimating the PMI of remains found in this context. Whole trotters (from which the cartilage was harvested) exhibited decompositional changes including darkening of the dermis, skin slippage, liquefaction of soft tissues and complete skeletonisation. The decompositional changes to cartilage included a loss of cartilage covering articular facets, changes in colour and texture, formation of orthorhombic crystals, a change in surface pH and colonisation by bacteria. The bacteria found on the cartilage surface were in close proximity to the crystals and when cultured on a B-41 medium were found to precipitate crystals of the same morphology and chemical composition to those found on the cartilage surface. Three species of bacteria (Acinetobacter calcoaceticus, Acinetobacter iwoffii and Grimontia hollisae) were identified based on gas chromatography–mass spectrometry (GC-MS) of their fatty acids and one species (Comamonas sp.) was identified by DNA analysis. Formation of crystals on goat and cow cartilage proved that this was not a porcine specific phenomenon. Human hair exhibited a gradual degradation over time but this was dependent on the characteristics of the burial environment. Decompositional changes included colonisation by fungi, erosions to the cortical surface and formation of tunnels and breaks to the hair shaft. Two fungal species (Aspergillus fumigatus and Penicillium sp.) were identified based on DNA analysis of fungal ribosomal (rDNA) internally transcribed spacer (ITS) regions. The Penicillium sp. was linked with fungal tunnelling of hair. Bone exhibited little modification over time but changes were observed. These included a change in colour of the cortical surface, a change in colour and gradual loss of bone marrow and erosions, cracking and flaking of the cortical bone. Fungi were found to colonise both the bone marrow and bone surface. Whole piglets were buried to document the time period taken to reach skeletonisation. This data was used as a correction factor and combined with the bone results to give an overall time period for the decomposition changes observed. The results of this study suggest that the decompositional changes to cartilage could be used to determine the postmortem interval of buried remains. However, the degradation of hair and bone was too variable to be of use in this context.
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Indian hedgehog stimulates chondrocyte hypertrophic differentiation inendochondral bone formationLi, Jun January 2007 (has links)
published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy
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Untersuchungen an humanen chondrogenen Progenitorzellen aus späten Stadien der Osteoarthrose zu dem Migrationspotential und der Proliferationsfähigkeit ex vivo sowie zu den Auswirkungen einer Kultivierung in vitro auf das Genexpressionsmuster / The ability for migration and proliferation ex vivo of human chondrogenic progenitor cells from late stages of osteoarthritis and the effects of in vitro culture on gene-expressionPath, Jan Ragnar 18 May 2015 (has links)
Diese Arbeit untersucht die humanen chondrogenen Progenitorzellen (CPC) auf ihre Eignung für einen möglichen Therapieansatz der Osteoarthrose (OA). Hierbei zeigte sich, dass die CPC die Fähigkeit zur Migration sowohl in vitro als auch ex vivo besitzen. In vitro steigerte der Einsatz von TGF-ß3 die Migrationsrate, was von therapeutischem Nutzen sein könnte. Die CPC wanderten ex vivo ebenso in Knorpelgewebe aus makroskopisch unauffälligen Bereichen wie in an den Hauptdefekt angrenzendes Gewebe ein. Mit der Versuchsdauer nahm die Einwanderungstiefe zu, wobei dies im unauffällig erscheinenden Knorpel ausgeprägter war und hier auch größere Eindringtiefen gemessen wurden. Dies spricht für eine Zielzone, die die CPC anstreben und die im Knorpel neben dem Hauptdefekt früher erreicht wird. Bezüglich der Migrationsgeschwindigkeit weisen die CPC eine Heterogenität auf, verglichen mit anderen Zelltypen im menschlichen Organismus ist sie insgesamt allerdings gering. Über die gesamte Versuchsdauer ließen sich in beiden Gewebetypen proliferationsfähige CPC nachweisen. Eine Genexpressionsanalyse zeigte, dass die CPC dedifferenzierten Chondrozyten am ähnlichsten sind und eine kurzfristige Kultivierung sie kaum beeinflusst. Die Transfektion veränderte die Ausrichtung der CPC nicht, sondern führte vornehmlich zu einer vorübergehenden generellen Herabregulierung der Genexpression. Die Ergebnisse zeigen, dass ein therapeutischer Einsatz der CPC möglich wäre, da die Zellen auf einfache und komplikationsarme Weise gewonnen und kultiviert werden können. Zudem eröffnen die ausgeprägten migratorischen Fähigkeiten der CPC einfache Applikationswege, z. B. im Rahmen einer intraartikulären Injektion oder Arthroskopie. Ebenso viel versprechend ist die Erkenntnis, dass die Zellen innerhalb des Gewebes vital bleiben und so an einer Regeneration teilhaben könnten. Vor einer zukünftigen Anwendung der CPC bleiben allerdings noch einige Fragen zu beantworten. So ist die Rolle der CPC in der Pathogenese der OA noch nicht vollständig geklärt. Außerdem bleibt offen, wie lange die CPC im Gewebe vital bleiben und ob sie in der Lage sind, im Gewebe hyaline Knorpel-EZM herzustellen. Die Ergebnisse dieser Arbeit geben daher Anlass für weitere Forschung auf diesem Gebiet.
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CLINICAL AND FUNCTIONAL ASSESSMENT FOLLOWING AUTOLOGOUS CHONDROCYTE IMPLANTATION TO THE KNEE: THE ROLE OF PATIENT REPORTED OUTCOMES, PERFORMANCE BASED ASSESSMENT, AND RESPONSE SHIFTHoward, Jennifer Sebert 01 January 2011 (has links)
Autologous chondrocyte implantation (ACI) is a cell based therapy for the treatment of articular cartilage defects. Numerous studies have reported outcomes following ACI using a variety of patient reported outcomes (PROs), but no clear recommendations exist regarding which PRO is the most responsive to changes following ACI. Few studies have documented changes in performance based assessments (PBAs) following ACI. Response shift theory proposes that residual changes in self-report measures occur over time. Failing to account for response shift may result in over or under reporting of outcomes from which clinical decisions are made. The purposes of this dissertation were 1) review the literature concerning ACI outcomes to determine the responsiveness of PROs to changes in self-reported function following ACI, 2) evaluate the reliability of PBAs among ACI patients, 3) develop a descriptive timeline for the return of function 1 year following ACI using both PROs and PBAs, and 4) utilize PROs and PBAs to evaluate patients undergoing ACI for evidence of response shift.
All PRO and PBA measures were collected preoperatively and 3, 6, and 12 months postoperatively. A retrospective then-test PRO evaluation of function prior to surgery was completed at 6 and 12 months. Response shift was calculated by subtracting the original pre-test score from the then-test score.
A systematic review and meta-analyses of existing ACI outcome studies resulted in the recommendation of the International Knee Documentation Committee Subjective Knee Form (IKDC) and Lysholm Knee Scale as highly responsive PROs among ACI patients of varying activity levels. Despite significant increases in PRO scores as early as 6 months following ACI, improvement in PBAs at 12 months following ACI were limited to stride length, walking speed, and step-up force. Finally, no evidence of a group level effect for response shift was observed. These results support the validity of traditional pre-test/post-test research designs with no need to account for response shift when evaluating treatment effects of ACI on the group level. However, the Western Ontario and McMasters University Osteoarthritis Index (WOMAC) did show evidence of a measurable response shift on a patient by patient basis.
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SERUM CARTILAGE OLIGOMERIC MATRIX PROTEIN: A BIOMARKER FOR ACUTE ARTICULAR CARTILAGE DAMAGEHoch, Johanna M. 01 January 2012 (has links)
Bone bruise lesions (BBL) are documented on MRIs diagnosing acute knee ligament injury (AKLI). Recent evidence has indicated that a majority of patients that sustain an AKLI, especially anterior cruciate ligament (ACL) knee injury, will develop post-traumatic osteoarthritis (PTOA) 10-20 years following injury. It has been proposed that the initial damage sustained to the articular cartilage overlying BBL causes a cascade of events that may result in PTOA.
Researchers have proposed a modification to treatment protocols for more severe BBL, or have stressed the need for the development of protective therapies to protect the articular cartilage. However, there are limited tools available to evaluate the clinical outcome of articular cartilage overlying BBL. Furthermore, damage to the cartilage overlying BBL may be different according to differing BBL severities. Therefore, the use of a cartilage degradation biomarker, serum cartilage oligomeric matrix protein (sCOMP) and the use of a BBL severity classification system may be useful to determine if differences exist between patients with and without BBL, and with differing BBL severities.
The purpose of this dissertation was to investigate the utility of sCOMP as a biomarker for acute articular cartilage damage. The purposes of these studies were to determine the inter and intraday reliability of this marker, to document sCOMP longitudinally in collegiate athletes and following AKLI, and to determine if differences in sCOMP and self-reported pain and function exist for patients with and without BBL, and differing BBL following AKLI.
The results of these studies indicated sCOMP measures had strong inter and intraday reliability. Additionally, exercise does seem to influence sCOMP levels; however, these elevations may not be clinically meaningful. Furthermore, sCOMP levels were not different between patients with BBL and without, and between differing BBL severities. The results of these studies support the use of a BBL severity classification for future research studies in order to further elucidate the outcomes of these lesions.
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Development of Cartilage-Derived Matrix Scaffolds via Crosslinking, Decellularization, and Ice-TemplatingRowland, Christopher January 2015 (has links)
<p>Articular cartilage is a connective tissue that lines the surfaces of diarthrodial joints; and functions to support and distribute loads as wells as facilitate smooth joint articulation. Unfortunately, cartilage possesses a limited capacity to self-repair. Once damaged, cartilage continues to degenerate until widespread cartilage loss results in the debilitating and painful disease of osteoarthritis. Current treatment options are limited to palliative interventions that seek to mitigate pain, and fail to recapitulate the native function. Cartilage tissue engineering offers a novel treatment option for the repair of focal defects as well as the complete resurfacing of osteoarthritic joints. Tissue engineering combines cells, growth factors, and biomaterials in order to synthesize new cartilage tissue that recapitulates the native structure, mechanical properties, and function of the native tissue. In this endeavor, there has been a growing interest in the use of scaffolds derived from the native extracellular matrix of cartilage. These cartilage-derived matrix (CDM) scaffolds have been show to recapitulate the native epitopes for cell-matrix interactions as well as provide entrapped growth factors; and have been shown to stimulate chondrogenic differentiation of a variety of cell types. Despite the potent chondroinductive properties of CDM scaffolds, they possess very weak mechanical properties that are several orders of magnitude lower than the native tissue. These poor mechanical properties lead to CDM scaffolds succumbing to cell-mediated contraction, which dramatically and unpredictably alters the size and shape of CDM constructs. Cell-mediated contraction not only prevents the fabrication of CDM constructs with specific, pre-determined dimensions, but also limits cellular proliferation and metabolic synthesis of cartilage proteins. This dissertation utilized collagen crosslinking techniques as well as ice-templating in order to enhance the mechanical properties of CDM scaffolds and prevent cell-mediated contraction. Furthermore, the decellularization of CDM was investigated in order to remove possible sources of immunogenicity. This work found that both physical and chemical crosslinking techniques were capable of preventing cell-mediated contraction in CDM scaffolds; however, the crosslinking techniques produced distinct effects on the chondroinductive capacity of CDM. Furthermore, the mechanical properties of CDM scaffolds were able to be enhanced by increasing the CDM concentration; however, this led to a concomitant decrease in pore size, which limited cellular infiltration. The pore size was able to be rescued through the use of an ice-templating technique that led to the formation of large aligned grooves, which enabled cellular infiltration. Additionally, a decellularization protocol was developed that successfully removed foreign DNA to the same order of magnitude as clinically approved materials, while preserving the native GAG content of the CDM, which has been shown to be critical in preserving the mechanical properties of the CDM. Altogether, this body of work demonstrated that dehydrothermal crosslinking was best suited for maintaining the chondroinductive capacity of the CDM, and given the appropriate scaffold fabrication parameters, such as CDM concentration and ice-templating technique, dehydrothermal treatment was able to confer mechanical properties that prevented cell-mediated contraction. To emphasize this finding, this work culminated in the fabrication of an anatomically-relevant hemispherical scaffold entirely from CDM alone. The CDM hemispheres not only supported chondrogenic differentiation, but also retained the original scaffold dimensions and shape throughout chondrogenic culture. These findings illustrate that CDM is a promising material for the fabrication of tailor-made scaffolds for cartilage tissue engineering.</p> / Dissertation
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Identification des petits protéoglycanes riches en leucine comme nouveaux substrats de la MMP-13 dans le cartilage humain et caractérisation de leur site de clivageMonfort Faure, Jordi January 2005 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
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Mesenchymal Stem Cell Constructs for Repair of Focal Cartilage Defects in an Ovine ModelSomerson, Jeremy 28 November 2016 (has links) (PDF)
Focal cartilage defects (FCD) of the knee joint remain a difficult area of treatment for orthopaedic surgeons, as they often progress to generalized osteoarthritis (OA). Osteochondral autograft transfer (OAT) to the damaged cartilage area has shown promise, but this has been associated with pain and bleeding at the site of graft harvest. The use of mesenchymal stem cells (MSCs) in a matrix to regenerate articular cartilage has been proposed. This work describes a prospective case-control series comparing OAT with a novel, MSC-seeded scaffold graft in the stifle joints of healthy merino sheep. The triphasic grafts were composed of a beta-tricalcium phosphate osseous phase, an intermediate activated plasma phase and a collagen I hydrogel cartilage phase.
The osseous and cartilage phases were seeded with autologous MSCs.
All sheep underwent creation of a full-thickness, 4.0 mm diameter FCD (n=20) followed by six weeks of unrestricted activity, allowing the defects to degenerate naturally. At six weeks, half of the lesions were treated with OAT and half with the triphasic engineered grafts.
At 6-month and 12-month follow-up, no significant differences were noted between groups with regard to overall histological scores. Macroscopic and biomechanical analysis at 12 months showed no significant differences between groups. In summary, autologous MSC-seeded implants showed comparable repair quality to OAT without the associated donor site morbidity.
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