Bone defects and fracture non-unions remain a substantial challenge for clinicians due to a high occurrence of delayed union or non-union requiring surgical intervention. The current grafting procedures used to treat these injuries have many limitations and further long-term complications associated with them. This has resulted in research efforts to identify graft substitution therapies that are able to repair and replace tissue function. Many of these tissue engineered products include the use of growth factors to induce cell differentiation, migration, proliferation, and/or matrix production. However, current growth factor delivery methods are limited by poor retention of growth factors upon implantation resulting in low bioactivity. These limiting factors lead to the use of high doses and frequent injections, putting the patients at risk for adverse effects. The goal of this work was to develop and evaluate the efficacy of BMP-2 delivery systems to improve bone regeneration. We examined two approaches for delivery of BMP-2 in this work. First, we evaluated the use of a self-assembling lipid microtube system for the sustained delivery of BMP-2. We determined that sustained delivery of BMP-2 from the lipid microtube system was able to enhance osteogenic differentiation compared to empty microtubes, however did not demonstrate a significant advantage compared to a bolus BMP-2 dose in vitro. Second, we developed and assessed the functionality of an affinity-based system to sequester BMP-2 at the implant site and retain bioactivity by incorporating heparin within a collagen matrix. Incorporation of heparin in the collagen matrix improved BMP-2 retention and bioactivity, thus enhancing cell-mediated mineralized matrix deposition in vitro. Lastly, the affinity-based BMP-2 delivery system was evaluated in a challenging in vivo bone repair model. Delivery of pre-bound BMP-2 and heparin in a collagen matrix resulted in new bone formation with mechanical properties not significantly different to those of intact bone. Whereas delivery of BMP-2 in collagen or collagen/heparin matrices had similar volumes of regenerated mineralized tissue but resulted in mechanical properties significantly less than intact bone properties. The work presented in this thesis aimed to address parameters currently preventing optimal performance of protein therapies including stability, duration of exposure, and localization at the treatment site. We were able to demonstrate that sustained delivery of BMP-2 from lipid microtubes was able to induce osteogenic differentiation, although this sustained delivery approach was not significantly advantageous over a bolus dose. Additionally, we demonstrated that the affinity-based system was able to improve BMP-2 retention within the scaffold and in vitro activity. However, in vivo implantation of this system demonstrated that only delivery of pre-complexed BMP-2 and heparin resulted in regeneration of bone with mechanical properties not significantly different from intact bone. These results indicate that delivery of BMP-2 and heparin may be an advantageous strategy for clinically challenging bone defects.

Non-healing bone defects have a significant socioeconomic impact in the U.S. with approximately 600,000 bone grafting procedures performed annually. Autografts and allografts are clinically the most common treatments; however, autologous donor bone is in limited supply, and allografts often have poor mechanical properties. Therefore, tissue engineering and regenerative medicine strategies are being developed to address issues with clinical bone grafting. The overall objective of this work was to develop bone tissue engineering strategies that enhance healing of orthotopic defects by targeting specific osteogenic cell signaling pathways. The general approach included the investigation of two different tissue engineering strategies, which both focused on directed osteoblastic differentiation to promote bone formation. In the first cell-based strategy, we hypothesized that constitutive overexpression of the osteoblast-specific transcription factor, Runx2, in bone marrow stromal cells (BMSCs) would promote orthotopic bone formation in vivo. We tested this hypothesis by delivering Runx2-modified BMSCs on synthetic scaffolds to critically-sized defects in rats. We found that Runx2-modified BMSCs significantly increased orthotopic bone formation compared to empty defects, cell-free scaffolds and unmodified BMSCs. This gene therapy approach to bone regeneration provides a mineralizing cell source which has clinical relevance. In the second biomaterial-based strategy, we hypothesized that incorporation of the collagen-mimetic peptide, GFOGER, into synthetic bone scaffolds would promote orthotopic bone formation in vivo without the use of cells or growth factors. We tested this hypothesis by passively adsorbing GFOGER onto poly-caprolactone (PCL) scaffolds and implanting them into critically-sized orthotopic defects in rats. We found that GFOGER-coated scaffolds significantly increased bone formation compared to uncoated scaffolds in a dose dependent manner. Development of this cell-free strategy for bone tissue engineering provides an inexpensive therapeutic alternative to clinical bone defect healing, which could be implemented as a point of care application. Both strategies developed in this work take advantage of specific osteoblastic signaling pathways involved in bone healing. Further development of these tissue engineering strategies for bone regeneration will provide clinically-relevant treatment options for healing large bone defects in humans by employing well-controlled signals to promote bone formation and eliminating the need for donor bone.

<p>Polysaccharides constitute an important source of raw materials for the packaging industry today. Polysaccharides have good natural barrier properties which are necessary for packaging films. Cellulose is the forerunner among renewable polymers for such applications. Hemicelluloses represent a new interesting breed of barrier materials. We have chosen to work with the hemicellulose O-acetyl-galactoglucomannan (AcGGM). The high water solubility of this particular hemicellulose extracted from process waters is both an advantage and a limiting factor. However, through the right modification, the water sensitivity of AcGGM can be regulated.</p><p>This thesis presents four ways to modify AcGGM: (i) benzylation, (ii) plasma surface treatment followed by styrene addition, (iii) vapor-phase (VP) surface grafting with styrene, and (iv) lamination of an unmodified film with a benzylated material. The most important methods of analysis of the films produced include contact angle measurement, dynamic mechanical analysis under moisture scan, and oxygen gas permeability measurement.</p><p>It was found that unmodified AcGGM films have low oxygen permeability at intermediate relative humidity (50 % RH) and good dynamic mechanical properties over a wider humidity range. Films of benzylated material (BnGGM) exhibited a decrease in oxygen permeability at lower humidity but showed better tolerance to higher humidities and indicated better dynamic mechanical behavior than AcGGM films. Lamination proved to be the most promising technique of modification, combining the good gas barrier properties of AcGGM films with the moisture-insensitivity of the BnGGM films.</p>

Die vorliegende Arbeit beschreibt verschiedene Synthesewege zur Oberflächenmodifizierung silikatischer Partikel mit 1,3-Divinylimidazolidin-2-on (Bisvinylethylenharnstoff, BVH) und Vinylformamid (VFA), z. B. radikalische Pfropfpolymerisationen („grafting from/to“) und radikalisch vernetzende Copolymerisation von VFA mit BVH. Es werden Versuche zur sauren Verseifung der Formamidgruppen und zur Charakterisierung der gebildeten Polyvinylamin (PVAm)/Kieselgel Hybridmaterialien vorgestellt. Die Eigenschaften der PVAm/Kieselgel Hybridmaterialien sind je nach zugrundeliegendem Polymerisationsprozeß verschieden und werden mittels 13 C{1 H}-CP-MAS-NMR-Spektroskopie, Zetapotentialmessungen, SANS, Quelltests und rheologischen Methoden untersucht. Die kationische Oberflächenpolymerisation von BVH unter Verwendung des Initiatorsystems Triphenylmethylchlorid/Kieselgel wird ausführlich erläutert. Die Struktur der resultierenden Hybridmaterialien wird mittels quantitativer Elementaranalyse, 13 C{1 H}-CP-MAS-NMR-, DRIFT- und ESCA- Spektroskopie, sowie TGA und Zetapotentialmessungen untersucht. Die Anwendung der Poly-BVH/KG Hybridmaterialien zur Adsorption von Schwermetallsalzen wird am Beispiel der Adsorption von CuCl2, CoCl2, CoI2 und FeCl3 beschrieben. Dabei werden der Einfluß des Polymergehaltes der Hybride und des Metallanions auf die adsorbierte Sättigungsstoffmenge diskutiert. Eine quantitative Beschreibung der Adsorption nach dem LANGMUIR-Modell wird gegeben, welche in einem Vorschlag für die Wechselwirkung zwischen Hybridmaterial und Metallsalz mündet. Mit der Adsorption von Goldnanoclustern an Poly-BVH/Kieselgel Hybriden wird eine weitere Möglichkeit der Modifizierung und des Aufbaus von funktionellen Multischichtsystemen vorgestellt.

Το ερευνητικό πεδίο της παρούσας διδακτορικής διατριβής ήταν η οστεοενσωμάτωση οστικών εμφυτευμάτων. Οι βασικοί στόχοι της έρευνας αφορούσαν την κατανόηση του ρόλου τριών βασικών ρυθμιστικών παραγόντων της διαδικασίας οστεοενσωμάτωσης, δηλαδή της μηχανικής φόρτισης, της επιφανειακής τοπογραφίας και της επιφανειακής (βιο)χημείας, καθώς και τη μελέτη του βασικότερου λόγου αστοχίας των ορθοπεδικών εμφυτευμάτων, της παραγωγής μικροσωματιδίων προϊόντων φθοράς τους. / -

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Contents 1. Introduction 7 1.1. The aim of the study 8 1.2. Tasks of the study 9 1.3. The scientific novelty and originality of the study 9 1.4. Practical importance of the study 9 2. Material and methods 11 2.1. The contingent of studied patients 11 2.2. Methods 12 2.2.1.Coronary artery bypass grafting 12 2.2.2. The used equipment 12 2.2.3. The method of myocardial SPECT performance 12 2.2.4. Evaluation of myocardial SPECT 13 2.3. Statistical analysis of data 15 3. Results 16 3.1. The evaluation of influence of left ventricular ejection fraction on postoperative changes of perfusion of revascularized myocardium 16 3.2. The evaluation of influence of left ventricle ejection fraction on postoperative changes of contraction of revascularized myocardium 18 3.3. The evaluation of influence of collaterals on postoperative changes of perfusion of revasculized myocardium 19 3.4. The evaluation of influence of collaterals on postoperative changes of contraction of revasculized myocardium 21 3.5. The prediction of postoperative myocardial perfusion and contraction 23 3.6. The identification of period of the maximal recovery of myocardial perfusion and contraction after surgical revascularization 27 4. Conclusions 29 5. List of publications 30 6. Summary in Lithuanian 31 7. Autobiography 34 1. Introduction Heart and blood vessels diseases, the most common of which is coronary artery disease (CAD), are the leading causes of death and disability of middle-aged and elderly... [to full text]

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For millennia, scions have been grafted onto dwarfing apple rootstocks to reduce final tree size. However, it is unclear how scion architecture is first modified by the dwarfing apple rootstock, the time from grafting when this occurs and the endogenous hormonal signalling mechanisms that may cause the initial modifications in growth that then define the future architecture of the scion. In this study, the dwarfing (M.9) rootstock significantly decreased the mean total shoot length and node number of ‘Royal Gala’ apple scions by the end of the first year of growth from grafting when compared with rootstock(s) of greater vigour (MM.106, M.793 and a ‘Royal Gala’ rootstock control). Similarly, the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA) applied to the stem of vigorous rootstocks significantly decreased mean total shoot length and node number of the scion, and the architectural changes imposed were generally similar to those imposed by M.9. For example, both treatments decreased the mean length and node number of the primary shoot, reduced the formation of secondary axes on the primary shoot and caused a greater proportion of primary and secondary shoots (if present) to terminate growth early. Decreased formation of secondary axes imposed by both treatments was reversed by applying the cytokinin benzylaminopurine (BAP) repeatedly to the scion, whilst applications of gibberellins (GA4+7) reduced the proportion of primary and secondary shoots that terminated growth early, therefore increasing the final mean length and node number of these shoot types. Both M.9 and NPA also significantly decreased the final mean dry mass and length of the root system. Given these general similarities, it is proposed that the basipetal IAA signal is of central importance in rootstock-induced scion dwarfing, and that a shoot/root/shoot signalling mechanism may exist whereby the stem tissue of the M.9 rootstock decreases the basipetal transport of IAA to the root during summer, thereby decreasing root growth and the amount of rootproduced cytokinin and gibberellin transported to scion. Reduced amounts of cytokinin transported to the scion may decrease branching, whilst reduced amounts of gibberellins may decrease the duration for which a large proportion of primary and secondary shoots grow. Analysis of endogenous hormones for newly grafted composite ‘Royal Gala’ apple trees on rootstocks of different vigour provided some additional support for these ideas. It is recommended that future studies elucidate what unique properties of the M.9 bark act to restrict IAA transport, whilst it is concluded that gene(s) regulating rootstock-induced scion dwarfing are likely to control processes within the rootstock that modify the metabolism of IAA, its basipetal transport and the subsequent synthesis of root-produced vigour-inducing hormones including cytokinins and gibberellins.

Thesis--København Tandlægehøjskole. / Summary in Danish. Bibliography: p. 179-194.