Specific objective: Guided tissue regeneration (GTR) aims to regenerate the lost attachment apparatus caused by periodontal disease through the use of a barrier membrane. For the GTR procedures to be successful, barrier membranes are required to be present at the surgical site for an extended period of time (weeks to months). Synthetic membranes have the advantage of prolonged presence in a wound site; however, they do not actively contribute to wound healing. Biologic membranes are recognized by the host tissue and participate in wound healing but have the disadvantage of early resorption. Therefore, the goal of this study is to create and characterize a hybrid barrier membrane that contains biologically active fibrin matrix within a synthetic polymeric electrospun scaffold. Method: Fibrin matrices and fibrin-incorporated electrospun scaffold were created from fresh frozen plasma at three different centrifugation conditions 400g for 12 minutes, 1450g for 15 minutes and 3000g for 60 minutes. Following centrifugation, half of the membranes were crosslinked with 1% genipin. Biological stability of these scaffolds was evaluated by resistance to trypsin while their mechanical properties were characterized by MTS Bionix Uniaxial Tensile Testing System. Continuous data was analyzed by ANOVA to detect differences between groups (p=0.05). Results: The addition of an electrospun scaffold to the fibrin matrix led to improvements in the mechanical properties as evidenced by an increase in the modulus (p<0.0001), strain at break (p<0.0001) and energy to break (p<0.0001). The effect of crosslinking was marginal but not statistically significant to the mechanical properties of fibrin matrices or the fibrin incorporated scaffold. However, crosslinking did significantly increase resistance against enzymatic degradation by trypsin (p<0.0001). Lastly, centrifugation speeds at 400g and 1450g showed similar mechanical properties and biologic stability; meanwhile 3000g negatively impacted the properties of the scaffold. Conclusion: Fibrin-incorporated electronspun scaffold exhibits enhanced mechanical and biologic stability compared to fibrin matrices alone. Moreover, crosslinking improves the biologic stability of the novel biomaterial. All these characteristics of the fibrin-incorporated matrix make this membrane a potentially more ideal barrier for GTR procedures to enhance periodontal wound healing.

This work is based on the Molpher SW project, which is client-server application aiding exploration of chemical space between two input molecules. Aim of master thesis was modify the current version of program to manage scaffold hopping technique. This technique represents molecule in a simplified way. The simpler molecule is called scaffold. First of all there was need to define seve- ral levels of granularity and for each level define morphing operators. Server was modified with respect for parallelization. Experimental exploration of chemical space with and without the new feature is part of this work too. Powered by TCPDF (www.tcpdf.org)

Philosophiae Doctor - PhD / The abnormal death of neurons in the central nervous system of individuals suffering from neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis, takes place by an intrinsic cell suicide programme known as apoptosis. This process is triggered by several stimuli, and consists of numerous pathways and cascades which lead to the death of neuronal cells. It is this multifactorial nature of neurodegenerative diseases that has over the years seen many researchers develop compounds that may serve as multi-target directed ligands (MTDLs) which could potentially confer neuroprotection by acting simultaneously on different receptors and target sites implicated in neurodegeneration. This study was aimed at developing MTDLs that may serve as neuroprotective agents by simultaneously (a) inhibiting N-methyl D-aspartate receptors (NMDAR) and blocking L-type voltage gated calcium channels (VGCC) thus regulating the Ca2+ influx mediated excitotoxic process; (b) inhibiting the monoamine oxidase enzymes A and -B (MAO-A/B) thus allowing increase in dopamine levels in the central nervous system and reducing the levels of the highly oxidative products produced by the activity of these enzymes; (c) possessing anti-apoptotic activity to halt the neuronal cell death process. In designing the compounds we focused on the structures of rasagiline and selegiline, two well-known MAO-B inhibitors and proposed neuroprotective agents, and of NGP1-01, a known VGCC blocker and NMDAR antagonist. The first series of compounds (reported in research article 1, Chapter 3), comprised polycyclic propargylamine and acetylene derivatives. Compounds 12, 15 and 16 from this series showed promising VGCC and NMDA receptor channel inhibitory activity ranging from 18 % to 59 % in micromolar concentrations, and compared favourably to the reference compounds. In the MAO-B assay, compound 10 exhibited weak MAO-B inhibition of 73.32 % at 300 μM. The rest of the series showed little to no activity on these target sites, despite showing significant anti-apoptotic activity. This suggested the compounds in this series to be exhibiting their neuroprotective action through some other mechanism(s) unexplored in this study.

Uppsatsnivå: C

Electrospinning is a process by which micro and nanofibrous scaffolds can be easily fabricated to mimic structures such as the extracellular matrix of bone. A number of materials have been used to fabricate such scaffolds making the process an extremely versatile tool in the field of bone tissue engineering. Many scaffolds however are hydrophobic, leading to poor cellular attachment and proliferation, whilst the actual process of electrospinning is highly variable, producing irregular scaffolds that can ultimately influence cell invasion and differentiation. The focus of this thesis was to address the issues of poor biocompatibility and irregular scaffold production in three commonly used polymers each with different mechanical properties and degradation profiles. Poly (ε-caprolactone) (PCL), polyethylene terephthalate (PET) and poly lactic-co-glycolic acid (PLGA) were functionalised with surfactants in order to improve the biocompatibility and osteoinductive properties of electrospun scaffolds, whilst electrospinning equipment was modified to improve uniformity of scaffold production. Reducing variables known to affect scaffold formation such as temperature and humidity through the use of an environmental stability cabinet improved the reproducibility of scaffolds. The introduction of a Faraday cage, a larger electrode and a negative mandrel potential also improved the quality and quantity of electrospun fibres collected. Lecithin was selected as an appropriate additive for both improving biocompatibility and uniformity of electrospun fibres as it is naturally occurring and induced dose dependent reductions in water contact angle, allowing tailored hydrophobicity. Through gravimetric determination of pore sizes coupled with mathematical modelling, the addition of lecithin was found to reduce both mean fibre diameter and pore size in all scaffolds, improving scaffold homogeneity. At low concentrations (i.e. 2 %) lecithin generally did not affect the mechanical properties of scaffolds, however significant improvements in tensile strength for PCL and nanoindentation for PET were evident, indicating these scaffolds remained suitably strong for bone regeneration purposes. Reduced hydrophobicity acted to improve cellular attachment of Saos-2 osteoblasts to polymers, whilst proliferation on all scaffolds was similar to TCP controls. Furthermore, lecithin incorporation induced osteoinduction, as bone marrow mesenchymal stem cells seeded on these hybrid scaffolds expressed upregulated gene expression for alkaline phosphatase, collagen 1, osteocalcin and osteopontin. In conclusion, these scaffolds, functionalised with lecithin, improve the homogeneity of fibrous mats allowing increased reproducibility and efficiency of the electrospinning process. Furthermore, the improved biocompatibility and osteoinductivity that lecithin presents, allows for the production of more suitable electrospun scaffolds in the field of bone tissue engineering.

Notre laboratoire a mis en évidence la présence de la protéine Proliferating Cell Nuclear Antigen (PCNA) localisée exclusivement dans le cytosol du neutrophile exerçant une activité anti-apoptotique. C'est une protéine dite "scaffolding" qui s'associe à de nombreuses protéines partenaires pour assumer ses fonctions. Dans des conditions physiologiques, la relocalisation du noyau vers le cytosol de PCNA s'effectue à la fin de différenciation granulocytaire. Mon projet de thèse a permis d'identifier que cette relocalisation cytoplasmique était dérégulée dans les cellules leucémiques contribuant au phénomène de survie exagérée et à la résistance à la chimiothérapie. Nous avons montré qu'il y avait une augmentation très significative de PCNA cytoplasmique dans le cytosol des cellules HL-60 rendues résistantes à la daunorubicine (HL-60R) résultant d'un export nucléaire actif, en comparaison aux cellules HL-60 sensibles (HL-60S). Dans ces cellules HL-60R, PCNA cytoplasmique interagit avec NAMPT, une protéine qui a un rôle clé dans la voie de la glycolyse leur conférant un avantage de survie des cellules.Enfin, dans le neutrophile, nous avons mis en évidence pour la première fois une association structurale et fonctionnelle entre la protéine cytosolique p47Phox de la NADPH oxydase et PCNA suggérant que ce dernier contrôle à la fois la survie et l'état de repos du neutrophile.PCNA est donc un facteur clé cytoplasmique dans la survie de plusieurs types cellulaires. Identifier ses mécanismes d'action dans le but de moduler ses partenaires s'avère être un axe de recherche très utile pour développer de nouveaux traitements thérapeutiques. / Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNI replication, has been described as a key element in survival of neutrophil, a non-proliferating cell. Without enzymatic activity this main function is to build a protein scaffold through the binding and functional coordination of its different partners. This relocation of PCNA from the nucleus into the cytoplasm occurs at the end of granulocytic differentiation. From our present findings, we propose new paradigm in which cytosolic PCNA builds a protein scaffold that dictates Acute Myeloid Leukemia (AML) cell survival by enhancing their glycolytic metabolism and in turn conferrinl chemotherapy resistance. We have demonstrated that daunorubicin-resistant HL-60 cells (HL-60R have a prominent cytosolic PCNA localization due to increased nuclear export compared to their sensitive counterpart. By interacting with nicotinamide phosphoribosyltransferase (NAMPT), protein involved in the NAD biosynthesis, PCNA coordinates the glycolysis pathway and survival especially in HL-60R cells.In neutrophil, we have also demonstrated a functional and structural interaction between a protein p47Phox: a cytosolic subunit of NADPH oxidase and PCNA which suggested that PCNA control the survival and maintain the resting state of neutrophils. PCNA is a key element involved in survival of different types of cells. Decipher the molecular mechanisms of PCNA to modulate its partners represent a promising avenue of research.

Clinical islet transplantation is a promising treatment for type 1 diabetes. It involves the transplantation of pancreatic islets, isolated from a donor, into the portal vein of a recipient in order to replace his/her dysfunctional islets. Though promising, islet implantation into the liver is greatly hindered by numerous problems, including mechanical stresses, inflammatory responses, exposure to high drug and toxin loads, as well as irretrievability. In order to address these concerns, investigation into alternative implant sites, such as the subcutaneous site, has intensified. Transplantation of islets within these extrahepatic sites is commonly met with three primary obstacles: 1) inadequate spatial distribution of the cells; 2) oxygen deficiency in the local environment; and 3) insufficient vascularization within and around the implant. Thus, the objective of this proposal is to engineer a superior bioartificial pancreas, a device combining novel biomaterials and insulin-secreting cells, by focusing on these critical issues, specifically how to best reduce islet aggregation, as well as increase oxygen delivery, both in the short term and long term. A highly macroporous silicone scaffold will be engineered to distribute the islets three-dimensionally, while not imparting diffusion resistances commonly encountered in microporous materials. Macroporous scaffolds will also permit vascular in-growth. In order to sustain oxygen levels at the moment of device implantation, a novel, oxygen generating disk, which relies on the decomposition of calcium peroxide, will be developed and incorporated alongside the scaffold to deliver short-term supplemental oxygen. Therefore, it is postulated that these bioactive scaffolds, which interact with islets on a spatial, chemical, and biological level, will improve the viability as well as the function of islets, both in vitro and in vivo, as compared to naked islets under extrahepatic conditions.

In scaffold-based tissue engineering, control of scaffold degradation turns out to be a critical issue for reliable clinical applications. Degradation in this thesis refers to mass loss. Most of the present control methods take the approach of scaffold material modification and/or scaffold work environment adjustment to address this issue. The latter can easily get to its limit, and the former is not promising in the in-vivo implementation. This thesis proposed a new approach to control of scaffold degradation, that is, closed-loop and real-time control. To realize this approach, this thesis has tackled three important problems, namely (1) effects on degradation, (2) modeling of degradation, and (3) real-time measurement of degradation. This thesis is grounded to a biomaterial called chitosan, as it is widely used for building scaffolds. For the first problem, a statistical experiment was designed and a factorial analysis was conducted. For the second problem, a combined empirical-based and probabilistic-based approach was taken. For the third problem, a prototype of a sensor, which is based on the concept of carbon nanotube (CNT) conductive polymer, was built and tested. This thesis concludes (1) a joint thermal and enzymatic effect is significant on chitosan degradation, (2) the model for chitosan degradation is accurate, and (3) real-time measurement of mass loss of scaffold by means of carbon nanotube film is feasible. The major contributions of this thesis are (i) the proposal of the concept of the closed-loop control of degradation, (ii) a finding that there is a significant conjoint thermal and enzymatic effect on chitosan degradation in terms of mass loss, and (iii) a prototype of the novel CNT (carbon nanotube) chitosan film sensor for real-time measurement of mass loss of the scaffold. The significance of these contributions is that they give us confidence to a full development of the closed-loop and real-time degradation control approach. This approach appears promising to bring forth a transformative impact to clinic applications of scaffold-based tissue regeneration.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 122-128). Also available on the World Wide Web.

Tissue engineering is an emerging field aimed to combine biological, engineering and material methods to create a biomimetic three dimensional (3D) environment to control cells proliferation and functional tissue formation. In such an artificial structural environment, a scaffold, made from biomaterial(s), plays an essential role by providing a mechanical support and biological guidance platform. Hence, fabrication of tissue scaffolds is of a fundamental importance, yet a challenging task, in tissue engineering. This task becomes more challenging if living cells need to be encapsulated in the scaffolds so as to fabricate scaffolds with structures to mimic the native ones, mainly due to the issue of process-induced cell damage. This research aims to develop novel methods to model the process of fabricating cell-encapsulated scaffolds and process-induced cell damage. Particularly, this research focuses on the scaffold fabrication process based on the dispensing-based rapid prototyping technique - one of the most promising scaffold fabrication methods nowadays, by which a 3D scaffold is fabricated by laying down multiple, precisely formed layers in succession. In the dispensing-based scaffold fabrication process, the flow behavior of biomaterials solution can significantly affect the flow rate of material dispensed, thus the structure of scaffold fabricated. In this research, characterization of flow behavior of materials was studied; and models to represent the flow behaviour and its influence on the scaffold structure were developed. The resultant models were shown able to greatly improve the scaffold fabrication in terms of process parameter determination. If cells are encapsulated in hydrogel for scaffold fabrication, cell density can affect the mechanical properties of hydrogel scaffolds formed. In this research, the influence of cell density on mechanical properties of hydrogel scaffolds was investigated. Furthermore, finite element analysis (FEA) of mechanical properties of scaffolds with varying cell densities was performed.The results show that the local stress and strain energy on cells varies at different cell densities. The method developed may greatly facilitate hydrogel scaffolds design to minimize cell damage in scaffold and promote tissue regeneration. . In the cell-encapsulated scaffold fabrication process, cells inevitably suffer from mechanical forces and other process-induced hazards. In such a harsh environment, cells deform and may be injured, even damaged due to mechanical breakage of cell membrane. In this research, three primary physical variables: shear stress, exposure time, and temperature were examined and investigated with regard to their effects on cell damage. Cell damage laws through the development phenomenal models and computational fluidic dynamic (CFD) models were established; and their applications to the cell-encapsulated scaffold fabrication process were pursued. The results obtained show these models and modeling methods not only allow one to optimize process parameters to preserve cell viability but also provide a novel strategy to probe cell damage mechanism in microscopic view.