Development of Craniofacial Bone Defect Reconstruction Implant Based on Fibre-Reinforced Composite with Photopolymerisable Resin Systems. Experimental studies in vitro and in vivo.

Reconstruction of defects in the craniomaxillofacial (CMF) area has mainly been based on bone grafts or metallic fixing plates and screws. Particularly in the case of large calvarial and/or craniofacial defects caused by trauma, tumours or congenital malformations, there is a need for reliable reconstruction biomaterials, because bone grafts or metallic fixing systems do not completely fulfill the criteria for the best possible reconstruction methods in these complicated cases. In this series of studies, the usability of fibre-reinforced composite (FRC) was studied as a biostable, nonmetallic alternative material for reconstructing artificially created bone defects in frontal and calvarial areas of rabbits. The experimental part of this work describes the different stages of the product development process from the first in vitro tests with resin-impregnated fibrereinforced composites to the in vivo animal studies, in which this FRC was tested as an implant material for reconstructing different size bone defects in rabbit frontal and calvarial areas. In the first in vitro study, the FRC was polymerised in contact with bone or blood in the laboratory. The polymerised FRC samples were then incubated in water, which was analysed for residual monomer content by using high performance liquid chromatography (HPLC). It was found that this in vitro polymerisation in contact with bone and blood did not markedly increase the residual monomer leaching from the FRC. In the second in vitro study, different adhesive systems were tested in fixing the implant to bone surface. This was done to find an alternative implant fixing system to screws and pins. On the basis of this study, it was found that the surface of the calvarial bone needed both mechanical and chemical treatments before the resinimpregnated FRC could be properly fixed onto it. In three animal studies performed with rabbit frontal bone defects and critical size calvarial bone defect models, biological responses to the FRC implants were evaluated. On the basis of theseevaluations, it can be concluded that the FRC, based on E-glass (electrical glass) fibres forming a porous fibre veil enables the ingrowth of connective tissues to the inner structures of the material, as well as the bone formation and mineralization inside the fibre veil. Bone formation could be enhanced by using bioactive glass granules fixed to the FRC implants. FRC-implanted bone defects healed partly; no total healing of defects was achieved. Biological responses during the follow-up time, at a maximum of 12 weeks, to resin-impregnated composite implant seemed to depend on the polymerization time of the resin matrix of the FRC. Both of the studied resin systems used in the FRC were photopolymerised and the heat-induced postpolymerisation was used additionally.

Avian Evolutionary Genomics: Studies of Ficedula Flycatchers

In this thesis, different genetic tools are used to investigate both natural variation and speciation in the Ficedula flycatcher system: pied (Ficedula hypoleuca) and collared (F. albicollis) flycatchers. The molecular evolution of a gene involved in postnatal body growth, GH, has shown high degree of conservation at the mature protein between birds and mammals, whereas the variation observed in its signal peptide seems to be adaptive in pied flycatcher (I & II). Speciation is the process by which reproductive barriers to gene flow evolve between populations, and understanding the mechanisms involved in pre- and post-zygotic isolation have been investigated in Ficedula flycatchers. The Z chromosome have been suggested to be the hotspot for genes involved in speciation, thus sequencing of 13 Z-linked coding genes from the two species in allopatry and sympatry have been conducted (III). Surprisingly, the majority of Z-linked genes seemed to be highly conserved, suggesting instead a potential involvement of regulatory regions. Previous studies have shown that genes involved in hybrid fitness, female preferences and male plumage colouration are sex-linked. Hence, three pigmentation genes have been investigated: MC1R, AGRP, and TYRP1. Of these three genes, TYRP1 was identified as a strong candidate to be associated with black-brown plumage variation in sympatric populations, and hence is a strong candidate for a gene contributing to pre-zygotic isolation (IV). In sympatric areas, where pied and collared flycatchers have overlapping breeding areas, hybridization sometimes occurs leading to the production of unfit hybrids. By using a proteomic approach a novel expression pattern in hybrids was revealed compared to the parental species (V) and differentially expressed proteins subsequently identified by sequence similarity (VI). In conclusion, the Z chromosome appears to play an important role in flycatcher speciation, but probably not at the coding level. In addition the novel expression patterns might give new insights into the maladaptive hybrids.

Validation Studies of Thermal-hydraulic Code for Safety Analysis of Nuclear Power Plants

This thesis gives an overview of the validation process for thermal hydraulic system codes and it presents in more detail the assessment and validation of the French code CATHARE for VVER calculations. Three assessment cases are presented: loop seal clearing, core reflooding and flow in a horizontal steam generator. The experience gained during these assessment and validation calculations has been used to analyze the behavior of the horizontal steam generator and the natural circulation in the geometry of the Loviisa nuclear power plant. The cases presented are not exhaustive, but they give a good overview of the work performed by the personnel of Lappeenranta University of Technology (LUT). Large part of the work has been performed in co-operation with the CATHARE-team in Grenoble, France. The design of a Russian type pressurized water reactor, VVER, differs from that of a Western-type PWR. Most of thermal-hydraulic system codes are validated only for the Western-type PWRs. Thus, the codes should be assessed and validated also for VVER design in order to establish any weaknesses in the models. This information is needed before codes can be used for the safety analysis. Theresults of the assessment and validation calculations presented here show that the CATHARE code can be used also for the thermal-hydraulic safety studies for VVER type plants. However, some areas have been indicated which need to be reassessed after further experimental data become available. These areas are mostly connected to the horizontal stem generators, like condensation and phase separation in primary side tubes. The work presented in this thesis covers a large numberof the phenomena included in the CSNI code validation matrices for small and intermediate leaks and for transients. Also some of the phenomena included in the matrix for large break LOCAs are covered. The matrices for code validation for VVER applications should be used when future experimental programs are planned for code validation.

Macroporous Scaffolds for Bone Engineering. Studies on Cell Culture and Ectopic Bone Formation

Bone engineering is a rapidly developing area of reconstructive medicine where bone inducing factors and/or cells are combined with a scaffold material to regenerate the structure and function of the original tissue. The aim of this study was to compare the suitability of different macroporous scaffold types for bone engineering applications. The two scaffold categories studied were a) the mechanically strong and stable titanium fiber meshes and b) the elastic and biodegradable porous polymers. Furthermore, bioactive modifications were applied to these basic scaffold types, and their effect on the osteogenic responses was evaluated in cell culture and ectopic bone formation studies. The osteogenic phenotype of cultured cell-scaffold constructs was heightened with a sol-gel derived titania coating, but not with a mixed titania-silica coating. The latter coating also resulted in delayed ectopic bone formation in bone marrow stromal cell seeded scaffolds. However, the better bone contact in early implantation times and more even bone tissue distribution at later times indicated enhanced osteoconductivity of both the coated scaffold types. Overall, the most promising bone engineering results were obtained with titania coated fiber meshes. Elastic and biodegradable poly(ε-caprolactone/D,L-lactide) based scaffolds were also developed in this study. The degradation rates of the scaffolds in vitro were governed by the hydrophilicity of the polymer matrix, and the porous architecture was controlled by the amount and type of porogen used. A continuous phase macroporosity was obtained using a novel CaCl2 • 6H2O porogen. Dynamic culture conditions increased cell invasion, but decreased cell numbers and osteogenicity, within the scaffolds. Osteogenic differentiation in static cultures and ectopic bone formation in cell seeded scaffolds were enhanced in composites, with 30 wt-% of bioactive glass filler.

Capability matrix – Identifying and evaluating the key capabilities of purchasing and supply management

Organizations gain resources, skills and technologies to find out the ultimate mix of capabilities to be a winner in the competitive market. These are all important factors that need to be taken into account in organizations operating in today's business environment. So far, there are no significant studies on the organizational capabilities in the field of PSM. The literature review shows that the PSM capabilities need to be studied more comprehensively. This study attempts to reveal and fill this gap by providing the PSM capability matrix that identifies the key PSM capabilities approached from two angles: there are three primary PSM capabilities and nine subcapabilities and, moreover, the individual and organizational PSM capabilities are identified and evaluated. The former refers to the PSM capability matrix of this study which is based on the strategic and operative PSM capabilities that complement the economic ones, while the latter relates to the evaluation of the PSM capabilities, such as the buyer profiles of individual PSM capabilities and the PSMcapability map of the organizational ones. This is a constructive case study. The aim is to define what the purchasing and supply management capabilities are and how they can be evaluated. This study presents a PSM capability matrix to identify and evaluate the capabilities to define capability gaps by comparing the ideal level of PSM capabilities to the realized ones. The research questions are investigated with two case organizations. This study argues that PSM capabilities can be classified into three primary categories with nine sub-categories and, thus, a PSM capability matrix with four evaluation categories can be formed. The buyer profiles are moreover identified to reveal the PSM capability gap. The resource-based view (RBV) and dynamic capabilities view (DCV) are used to define the individual and organizational capabilities. The PSM literature is also used to define the capabilities. The key findings of this study are i) the PSM capability matrix to identify the PSM capabilities, ii) the evaluation of the capabilities to define PSM capability gaps and iii) the presentation of the buyer profiles to identify the individual PSM capabilities and to define the organizational PSM capabilities. Dynamic capabilities are also related to the PSM capability gap. If a gap is identified, the organization can renew their PSM capabilities and, thus, create mutual learning and increase their organizational capabilities. And only then, there is potential for dynamic capabilities. Based on this, the purchasing strategy, purchasing policy and procedures should be identified and implemented dynamically.

Non-resorbable glass fibre-reinforced composite with porous surface as bone substitute material: Experimental studies in vitro and in vivo focused on bone-implant interface

The development of load-bearing osseous implant with desired mechanical and surface properties in order to promote incorporation with bone and to eliminate risk of bone resorption and implant failure is a very challenging task. Bone formation and resoption processes depend on the mechanical environment. Certain stress/strain conditions are required to promote new bone growth and to prevent bone mass loss. Conventional metallic implants with high stiffness carry most of the load and the surrounding bone becomes virtually unloaded and inactive. Fibre-reinforced composites offer an interesting alternative to metallic implants, because their mechanical properties can be tailored to be equal to those of bone, by the careful selection of matrix polymer, type of fibres, fibre volume fraction, orientation and length. Successful load transfer at bone-implant interface requires proper fixation between the bone and implant. One promising method to promote fixation is to prepare implants with porous surface. Bone ingrowth into porous surface structure stabilises the system and improves clinical success of the implant. The experimental part of this work was focused on polymethyl methacrylate (PMMA) -based composites with dense load-bearing core and porous surface. Three-dimensionally randomly orientated chopped glass fibres were used to reinforce the composite. A method to fabricate those composites was developed by a solvent treatment technique and some characterisations concerning the functionality of the surface structure were made in vitro and in vivo. Scanning electron microscope observations revealed that the pore size and interconnective porous architecture of the surface layer of the fibre-reinforced composite (FRC) could be optimal for bone ingrowth. Microhardness measurements showed that the solvent treatment did not have an effect on the mechanical properties of the load-bearing core. A push-out test, using dental stone as a bone model material, revealed that short glass fibre-reinforced porous surface layer is strong enough to carry load. Unreacted monomers can cause the chemical necrosis of the tissue, but the levels of leachable resisidual monomers were considerably lower than those found in chemically cured fibre-reinforced dentures and in modified acrylic bone cements. Animal experiments proved that surface porous FRC implant can enhance fixation between bone and FRC. New bone ingrowth into the pores was detected and strong interlocking between bone and the implant was achieved.

Studies on 68Ga-Based Agents for PET Imaging of Cancer and Inflammation

Studies on ⁶⁸Ga-Based Agents for PET Imaging of Cancer and Inflammation Positron emission tomography (PET) is based on the use of radiolabeled agents and facilitates in vivo imaging of biological processes, such as cancer. Because the detection of cancer is demanding and is often obscured by inflammation, there is a demand for better PET imaging agents. The aim was to preliminarily evaluate new PET agents for imaging cancer and inflammation using experimental models. ⁶⁸Ga-chloride and peptides, ⁶⁸Ga-labeled through 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), targeting matrix metalloproteinase-9 (MMP-9) were tested for tumor imaging. In addition, a ⁶⁸Ga-DOTA-conjugated peptide targeting vascular adhesion protein-1 (VAP-1), was tested for inflammation imaging. The ⁶⁸Ga-based imaging agents described here showed potential features by passing the essential in vitro tests, proceeding further to preclinical in vivo evaluation and being able to visualize the target. The target uptake and target-to-background ratios of ⁶⁸Ga-based agents were, however, not optimal. ⁶⁸Ga-chloride showed slow clearance caused by its binding to blood transferrin. In the case of ⁶⁸Ga-DOTA-peptides low in vivo stability and/or low lipophilicity led to too rapid blood clearance and urinary excretion. The properties of ⁶⁸Ga-labeled peptides are modifiable, as shown with matrix metalloproteinase-9 targeting ligands. In the conclusion of this PhD thesis, ⁶⁸Ga-based agents for PET imaging of cancer and inflammation could be applied in the development of drugs, earlier diagnostics and following-up of the efficacy of therapies.

Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

Studies on chemoenzymatic synthesis: Lipase-catalyzed acylation in multistep organic synthesis

In this thesis, biocatalysis is defined as the science of using enzymes as catalysts in organic synthesis. Environmental aspects and the continuously expanding repertoire of available enzymes have firmly established biocatalysis as a prominent means of chemo-, regio- and stereoselective synthesis. Yet, no single methodology can solve all the challenges faced by a synthetic chemist. Therefore, the knowledge and the skills to combine different synthetic methods are relevant. Lipases are highly useful enzymes in organic synthesis. In this thesis, an effort is being made to form a coherent picture of when and how can lipases be incorporated into nonenzymatic synthesis. This is attempted both in the literature review and in the discussion of the results presented in the original publications contained in the thesis. In addition to lipases, oxynitrilases were also used in the work. The experimental part of the thesis comprises of the results reported in four peer-reviewed publications and one manuscript. Selected amines, amino acids and sugar-derived cyanohydrins or their acylated derivatives were each prepared in enantio- or diastereomerically enriched form. Where applicable, attempts were made to combine the enzymatic reactions to other synthetic steps either by the application of completely separate sequential reactions with isolated intermediates (kinetic and functional kinetic resolution of amines), simultaneously occurring reactions without intermediate isolation (dynamic kinetic resolution of amino acid esters) or sequential reactions but without isolating the intermediates (hydrocyanation of sugar aldehydes with subsequent diastereoresolution). In all cases, lipase-catalyzed acylation was the key step by which stereoselectivity was achieved. Lipase from Burkholderia cepacia was a highly selective enzyme with each substrate category, but careful selection of the acyl donor and the solvent was important as well.

Experimental Studies on Non-Metallic Composite Bone Implants With a Special Reference to Staphylococcal Biofilm Infection

Non-metallic implants made of bioresorbable or biostable synthetic polymers are attractive options in many surgical procedures, ranging from bioresorbable suture anchors of arthroscopic surgery to reconstructive skull implants made of biostable fiber-reinforced composites. Among other benefits, non-metallic implants produce less interference in imaging. Bioresorbable polymer implants may be true multifunctional, serving as osteoconductive scaffolds and as matrices for simultaneous delivery of bone enhancement agents. As a major advantage for loading conditions, mechanical properties of biostable fiber-reinforced composites can be matched with those of the bone. Unsolved problems of these biomaterials are related to the risk of staphylococcal biofilm infections and to the low osteoconductivity of contemporary bioresorbable composite implants. This thesis was focused on the research and development of a multifunctional implant model with enhanced osteoconductivity and low susceptibility to infection. In addition, the experimental models for assessment, diagnostics and prophylaxis of biomaterial-related infections were established. The first experiment (Study I) established an in vitro method for simultaneous evaluation of calcium phosphate and biofilm formation on bisphenol-Aglycidyldimethacrylate and triethylenglycoldimethacrylate (BisGMA-TEGDMA) thermosets with different content of bioactive glass 45S5. The second experiment (Study II) showed no significant difference in osteointegration of nanostructured and microsized polylactide-co-glycolide/β-tricalcium phosphate (PLGA /β-TCP) composites in a minipig model. The third experiment (Study III) demonstrated that positron emission tomography (PET) imaging with the novel 68Ga labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) CD33 related sialic-acid immunoglobulin like lectins (Siglec-9) tracer was able to detect inflammatory response to S. epidermidis and S. aureus peri-implant infections in an intraosseous polytetrafluoroethylene catheter model. In the fourth experiment (Study IV), BisGMATEGDMA thermosets coated with lactose-modified chitosan (Chitlac) and silver nanoparticles exhibited antibacterial activity against S. aureus and P. aeruginosa strains in an in vitro biofilm model and showed in vivo biocompatibility in a minipig model. In the last experiment (Study V), a selective androgen modulator (SARM) released from a poly(lactide)-co-ε-caprolactone (PLCL) polymer matrix failed to produce a dose-dependent enhancement of peri-implant osteogenesis in a bone marrow ablation model.

Isolation and Characterization of Ovomucin - a Bioactive Agent of Egg White

The hen’s egg is a source of new life. Therefore, it contains many biologically active compounds. In addition to being a very nutritious food and also commonly used in the food industry due to its many techno-functional properties, the egg can serve as a source of compounds used as nutra-, pharmaand cosmeceuticals. One such interesting compound is ovomucin, an egg white protein responsible for the gel-like properties of thick egg white. Previous studies have indicated that ovomucin and ovomucin-derived peptides have several different bioactive properties. The objectives of the present study were to develop isolation methods for ovomucin, to characterize the structure of ovomucin, to compare various egg fractions as sources of ovomucin, to study the effects of various dissolving methods for ovomucin, and to investigate the bioactive properties of ovomucin and ovomucin-derived peptides. A simple and rapid method for crude ovomucin separation was developed. By using this method crude ovomucin was isolated within hours, compared to the 1-2 days (including a dialysis step) needed when using several other methods. Structural characterization revealed that ovomucin is composed of two subunits, α- and β-ovomucin, as egg white protein formerly called α1-ovomucin seemed to be ovostatin. However, it might be possible that ovostatin is associated within β- and α-ovomucin. This interaction could even have some effect on the physical nature of various egg white layers. Although filtration by-product fraction was a very prominent source of both crude and β-ovomucin, process development has reduced its amount so significantly that it has no practical meaning anymore. Thus, the commercial liquid egg white is probably the best option, especially if it generally contains amounts of β-ovomucin as high as were found in these studies. Crude ovomucin was dissolved both by using physical and enzymic methods. Although sonication was the most effective physical method for ovomucin solubilisation, colloid milling seemed to be a very promising alternative. A milk-like, smooth and opaque crude ovomucin suspension was attained by using a colloid mill. The dissolved ovomucin fractions were further tested for bioactive properties, and it was found that three dissolving methods tested produced moderate antiviral activity against Newcastle disease virus, namely colloid milling, enzymatic hydrolysis and a combination of sonicaton and enzymatic hydrolysis. Moreover, trypsin-digested crude ovomucin was found to have moderate antiviral activity against avian influenza virus: both subtype H5 and H7.