Characterization of Early Biofilm Formation and Physiology in \u3ci\u3eNeisseria gonorrhoeae\u3c/i\u3e

Many bacteria rely on the dynamics of their extracellular appendages to perform important tasks, like motility and biofilm formation. Interestingly, these dynamics have been linked to physiological responses in some pathogenic bacteria; therefore, it is important to understand more about the role of physical forces in bacteria. I used the causative agent of the human disease gonorrhea, Neisseria gonorrhoeae, as a model system to study the role of physical force on early biofilm formation. The advantage of this system is that cell-cell interactions are controlled by extracellular filaments called type IV pili (tfp). Tfp is composed of monomers that give bacteria the ability to produce a dynamic filament undergoing cycles of elongations and retractions, and thus to exert forces on their surroundings. Through experiments and modeling, I demonstrated that pilus interactions produce motility gradients in microcolonies potentially establishing a force gradient across the microcolonies. I was interested in testing the biological implications of those motility and force gradients, so I utilized an established genetic mutant, ∆pilT, which lacks the pilus retraction motor pilT (Merz, So, and Sheetz 2000). A ∆pilT mutant allowed us to measure physiological response in cells that do not produce retractive force from its pilus. I measured the level of gene expression of seven pilus-related genes in two backgrounds: WT and a pilus retraction-deficient mutant, ∆pilT. I found that some WT microcolonies express pilus-related genes in a heterogeneous fashion, while others are homogeneous. Spatiotemporal patterns in the microcolony are modified in a ∆pilT background. The presence or absence of retraction forces between bacteria have a profound impact on bacterial physiology: the WT and ∆pilT background do not survive in a classical static biofilm assay at the same rate. Together these results point toward a fundamental role for intracellular forces in shaping bacteria physiology. The work of biologists has been dominated by a biochemical perspective. Although biochemical processes, like metabolism and information transfer, are certainly essential in all hierarchical levels of life, there is growing evidence that physical forces may provide an alternate physiological mechanism. The introduction in Chapter 1 provides context for understanding the role of force pattern formation in multicellular structures, in the hopes to extend this line of thinking to microbial communities. The development of microbial communities relies on self-assembly of single cells. The development of Neisseria gonorrhoeae cellular aggregates rely exclusively on type IV pili interactions (Taktikos et al. 2015a). In Chapter 2 is a transcription of the publication where I explore the dynamics of the microcolonies (W. Pönisch et al. 2018a). We found that cells have differential motility depending where in the microcolony cells are located. Differential motility is a result of fewer pili-pili interactions on the perimeter of the microcolony, and more pili-pili interactions closer to the center. Therefore, due to frequency of pili-pili interactions, a gradient of motility produces heterogeneous behavior in the microcolony. To investigate whether heterogenous behavior is extended beyond motility, I investigated whether there is a connection between retraction force and the physiology of microcolonies. In Chapter 3 I used a quantitative approach to analyze seven pilus-related genes using fluorescent reporters. Using fluorescence and confocal microscopy, I quantified fluorescence intensity within space and time in microcolonies. Here, I provide evidence that physical intracellular cues in a three-dimensional bacterial aggregate provide context for spatial organization, since spatiotemporal patterning and survival in ∆pilT background are compromised in comparison to WT microcolonies. This suggests the important role PilT retraction force plays in regulating spatiotemporal patterning during early biofilm development. Lastly, in Chapter 4 I characterized some physical features of microcolonies. I measured the formation size and survival rates of microcolonies when exposed to a range of osmotic pressures. These experiments were motivated by my interest in understanding the native context of developing microcolonies. Microcolonies inhabit the viscous mucosal membranes of epithelial cells; therefore, I measured one aspect of the environmental effects of microcolony when exposed to similar osmotic pressure created by mucus. I also measured the plasticity of WT and ∆pilT microcolonies through squeezing microplate experiments. The overall aim of this work is to understand the role of physical force on microbial development. I largely focused on role of tfp forces on Neisseria gonorrhoeae microcolony formation. Characterizing gene expression in microcolonies provided key evidence for spatiotemporal heterogeneity in developing WT microcolonies. Heterogeneity was minimized without pilus retraction forces, which suggests that retraction forces play a role in the early development of biofilm formation

Engineering biological gradients

Biological gradients profoundly influence many cellular activities, such as adhesion, migration, and differentiation, which are the key to biological processes, such as inflammation, remodeling, and tissue regeneration. Thus, engineered structures containing bioinspired gradients can not only support a better understanding of these phenomena, but also guide and improve the current limits of regenerative medicine. In this review, we outline the challenges behind the engineering of devices containing chemical-physical and biomolecular gradients, classifying them according to gradient-making methods and the finalities of the systems. Different manufacturing processes can generate gradients in either in-vitro systems or scaffolds, which are suitable tools for the study of cellular behavior and for regenerative medicine; within these, rapid prototyping techniques may have a huge impact on the controlled production of gradients. The parallel need to develop characterization techniques is addressed, underlining advantages and weaknesses in the analysis of both chemical and physical gradients

SOLID-SHELL FINITE ELEMENT MODELS FOR EXPLICIT SIMULATIONS OF CRACK PROPAGATION IN THIN STRUCTURES

Crack propagation in thin shell structures due to cutting is conveniently simulated using explicit finite element approaches, in view of the high nonlinearity of the problem. Solidshell elements are usually preferred for the discretization in the presence of complex material behavior and degradation phenomena such as delamination, since they allow for a correct representation of the thickness geometry. However, in solid-shell elements the small thickness leads to a very high maximum eigenfrequency, which imply very small stable time-steps. A new selective mass scaling technique is proposed to increase the time-step size without affecting accuracy. New ”directional” cohesive interface elements are used in conjunction with selective mass scaling to account for the interaction with a sharp blade in cutting processes of thin ductile shells

Development of porous glass-fiber reinforced composite for bone implants. Evaluation of antimicrobial effect and implant fixation

Cranial bone reconstructions are necessary for correcting large skull bone defects due to trauma, tumors, infections and craniotomies. Traditional synthetic implant materials include solid or mesh titanium, various plastics and ceramics. Recently, biostable glass-fiber reinforced composites (FRC), which are based on bifunctional methacrylate resin, were introduced as novel implant solution. FRCs were originally developed and clinically used in dental applications. As a result of further in vitro and in vivo testing, these composites were also approved for clinical use in cranial surgery. To date, reconstructions of large bone defects were performed in 35 patients. This thesis is dedicated to the development of a novel FRC-based implant for cranial reconstructions. The proposed multi-component implant consists of three main parts: (i) porous FRC structure; (ii) bioactive glass granules embedded between FRC layers and (iii) a silver-polysaccharide nanocomposite coating. The porosity of the FRC structure should allow bone ingrowth. Bioactive glass as an osteopromotive material is expected to stimulate the formation of new bone. The polysaccharide coating is expected to prevent bacterial colonization of the implant. The FRC implants developed in this study are based on the porous network of randomly-oriented E-glass fibers bound together by non-resorbable photopolymerizable methacrylate resin. These structures had a total porosity of 10–70 volume %, of which > 70% were open pores. The pore sizes > 100 μm were in the biologically-relevant range (50-400 μm), which is essential for vascularization and bone ingrowth. Bone ingrowth into these structures was simulated by imbedding of porous FRC specimens in gypsum. Results of push-out tests indicated the increase in the shear strength and fracture toughness of the interface with the increase in the total porosity of FRC specimens. The osteopromotive effect of bioactive glass is based on its dissolution in the physiological environment. Here, calcium and phosphate ions, released from the glass, precipitated on the glass surface and its proximity (the FRC) and formed bone-like apatite. The biomineralization of the FRC structure, due to the bioactive glass reactions, was studied in Simulated Body Fluid (SBF) in static and dynamic conditions. An antimicrobial, non-cytotoxic polysaccharide coating, containing silver nanoparticles, was obtained through strong electrostatic interactions with the surface of FRC. In in vitro conditions the lactose-modified chitosan (chitlac) coating showed no signs of degradation within seven days of exposure to lysozyme or one day to hydrogen peroxide (H2O2). The antimicrobial efficacy of the coating was tested against Staphylococcus aureus and Pseudomonas aeruginosa. The contact-active coating had an excellent short time antimicrobial effect. The coating neither affected the initial adhesion of microorganisms to the implant surface nor the biofilm formation after 24 h and 72 h of incubation. Silver ions released to the aqueous environment led to a reduction of bacterial growth in the culture medium.Siirretty Doriast

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.Polymeereistä valmistettuihin ei-metallisiin komposiittiluuimplantteihin liittyvä kokeellinen tutkimus erityisesti liittyen biofilmiä muodostavaan stafylokokki -infektioon Synteettisistä polymeereistä valmistetut biohajoavat ja biostabiilit implantit ovat vaihtoehtoja metallisille implanteille useissa kirurgisissa toimenpiteissä. Biohajoavia implantteja voidaan käyttää tähystyskirurgiassa ommelankkureina tai biostabiileita kuitulujitteisia implantteja kalloluun korjauksessa. Polymeereistä valmistetut implantit häiritsevät kuvantamistutkimuksia vähemmän kuin tavanomaiset metalliset implantit. Biohajoavat polymeerit voivat myös toimia luun kasvua ohjaavana rakenteena ja samalla vapauttaa paikallisesti luun kasvua edistävää lääkettä. Myös biostabiileista kuitulujitteisista polymeereistä voidaan valmistaa kuormaa kantavia implantteja, joiden mekaanisia ominaisuuksia on säädelty luun kanssa samankaltaisiksi. Biomateriaalien kliiniseen käyttöön liittyy aina jonkinasteinen infektioriski. Vaikeimmin hoidettavat infektiot liittyvät bakteerikantojen kykyyn kiinnittyä implanttien pinnalle (biofilmin muodostuminen). Myös biohajoavien implanttien käytössä riittämätön uudisluun muodostuminen on todettu yhdeksi ongelmaksi. Tässä väitöskirjatutkimuksessa keskityttiin multifunktionaalisen implanttimallin tutkimukseen, jossa pyrkimyksenä oli saada parannettua implantin osteokonduktiivisuutta ja alentaa infektioalttiutta. Ensimmäisessä osatyössä kehitettiin menetelmä, jolla voidaan seurata biofilmin ja hydroksiapatiitin muodostumista bioaktiivisen lasin 45S5 sekä bisfenoli-Aglysidyylidimetakrylaatin ja trietyleeniglykolidimetakrylaatin (BisGMA-TEGDMA) seoksesta valmistetun yhdistelmämateriaalin pinnalla. Toisessa osatyössä todettiin, että luun muodostusta edistävän β-trikalsiumfosfaatin (β-TCP) partikkelikoolla (nano versus mikro) ei ollut merkittävää vaikutusta biohajoavan yhdistelmämateriaalin (valmistettu polylaktidiglysidyylin seospolymeeristä ja β-TCP:sta) kiinnittymiseen luuhun. Kolmannessa osatyössä todettiin, että positroniemissiotomografian (PET) uusi Siglec-9 -niminen kuvausmerkkiaine pystyy tunnistamaan stafylokokkien (S. epidermidis ja S. aureus kannat) aiheuttamat implantti-infektiot. Siglec-9 valmistettiin 68Ga-isotoopilla leimattuun 1,4,7,10-tetra-atsasyklododekaani-1,4,7,10- tetraetikkahappoon (DOTA). Implantti oli luun sisäinen katetrityyppinen ratkaisu, ja se oli valmistettu polytetrafluorietyleenistä (PTFE). Neljännessä osatyössä osoitettiin, että nanokoon hopeapartikkeleita sisältävä laktoosi-modifioitu kitosaani (Chitlac) -pinnoite estää bakteerien kasvua (S. aureus ja P. aeruginosa kannat) biostabiilin BisGMA-TEGDMA:sta valmistetun implantin pinnalla. Pinnoitteella ei ollut haitallisia vaikutuksia implantin kiinnittymiseen luuhun. Viimeisessä osatyössä todettiin, että biohajoavista polylaktidikaprolaktonin (PLCL) seospolymeerimatriisista valmistetusta implantista vapautuva anabolinen lääkeaine (selektiivinen androgeenimodulaattori, SARM) ei edistänyt uudisluun muodostumista paikallisesti luuydinablaatiomallissa.Siirretty Doriast

Functional Ceramic Coatings

Ceramic materials in the form of coatings can significantly improve the functionality and applications of other engineering materials. Due to a wide range of controllable features and various deposition methods, it is possible to create tailored substrate–coating systems that meet the requirements of modern technologies. Therefore, it is crucial to understand the relationships between the structures, morphology and the properties of ceramic coatings and expand the base of scientific knowledge about them. This book contains a series of fourteen articles which present research on the production and properties of ceramic coatings designed to improve functionality for advanced applications