Les stents urétéraux et les sondes de néphrostomie sont constitués de silicone ou de polyuréthane thermoplastique (TPU). Afin de limiter les risques infectieux lors de leur implantation, une modification topographique par création de pores permettrait de limiter l’adhésion des bactéries et de former des réservoirs pour une libération in situ de substances antiinfectieuses. Ce travail vise à préparer des surfaces en polymère ayant un motif poreux tubulaire ordonné. Une réplication en deux temps à partir de surfaces ordonnées poreuses d’oxyde d’aluminium (PAAO) préparées par 2 méthodes (double anodisation douce et double anodisation dure/douce) a été mise en œuvre pour reproduire le motif initial sur des surfaces en silicone et TPU. Pour le moule intermédiaire trois matériaux ont été testés (acrylonitrile butadiène styrène, polystyrène et résine polyacrylate). Les surfaces ont toutes été caractérisées par des techniques microscopiques et spectroscopiques. Les surfaces en PAAO préparées par double anodisation douce possédaient des pores d’environ 50 nm de diamètre et 100 nm de profondeur, alors que celles obtenues après mise au point de la méthode dure/douce étaient de taille supérieure, d’environ 125 nm de diamètre et ayant des profondeurs de quelques centaines de nanomètres. La surface du moule intermédiaire est constituée de picots. Une adhésion latérale de ceux-ci a été observée pour certaines conditions. La meilleure réplication du motif a été obtenue pour le TPU. Les surfaces ainsi obtenues pourront être utilisées et optimisées lors de l'étude ultérieure de l'adhésion du biofilm. / Ureteral stents and nephrostomy catheters are made of silicone or thermoplastic polyurethane (TPU). A topographical modification creating an ordered porous surface could limit the infectious risks during their implantation, by reducing bacterial adhesion and creating a loading platform from which anti-infectious compounds could be released.In this work, a two-steps replication method was used to create ordered porous polymer surfaces (silicone or TPU) using porous anodic aluminium oxide (PAAO) as master template. The PAAO surfaces were prepared by double mild or double hard/mild anodization. Three intermediate mould materials were tested (acrylonitrile butadiene styrene, polystyrene, polyacrylate resin). The polymer material (silicone or TPU) was then moulded onto the intermediate mould surfaces that possessed freestanding pillar arrays, to imprint pores. The obtained surfaces were characterized by microscopic and spectroscopic methods. The initial PAAO surfaces prepared by double mild anodization possessed pores of about 50 nm diameter and 100 nm depth, whereas those prepared after development of the double hard/mild anodization method were bigger, of about 125 nm diameter and several hundred nanometers deep. The intermediate mould structure possessed freestanding arrays, but instabilities (lateral adhesion) were noted for certain conditions. The best pattern replication was observed for TPU. In conclusion, these novel porous polymeric surfaces could be optimized and tested for an anti-biofilm effect.

Malgré les avancées thérapeutiques récentes, le pronostic des patients porteurs de métastases osseuses (MO) reste faible, ce qui incite à chercher des nouvelles stratégies thérapeutiques. Les chimiokines sont des acteurs majeurs de la réponse immune, et apparaissent comme des cibles potentielles de l’immunothérapie anti-cancéreuse. Nous avons recherché à définir si la chimiokine CX3CL1 pouvait représenter un axe thérapeutique efficace dans le contexte des MO. Pour cela nous avons développé des modèles murins de MO de cancer du rein et du poumon. Dans le modèle de MO de cancer du poumon, notre travail a démontré que l'expression de CX3CL1 inhibe la croissance tumorale. L’analyse transcriptomique des tumeurs a montré que CX3CL1 diminue (i) l’ostéloyse via un effet sur la triade OPG/RANKL/RANK (ii) l'expression de certains checkpoints, en faveur d’une réponse immune antitumorale. En revanche, dans le modèle de MO de cancer du rein, l’expression de CX3CL1 stimule le développement tumoral et l'ostéolyse via une action sur la triade OPG/RANKL/RANK et inhibe la réponse immune antitumorale via une augmentation de l'expression de certains checkpoints immunitaires. Les bisphosphonates (BPs) sont des agents utilisés pour le traitement des MO. Afin de réduire leurs effets indésirables, nous avons utilisé des ciments de phosphate de calcium (CPC), pour délivrer localement dans l’os des BPs (alendronate, ALN). Notre travail a mis en évidence que (i) ces ciments chargés en ALN relarguent en continue les BPs, (ii) le relarguage d’ALN est efficace pour induire des effets cytotoxiques et pro-apoptotiques vis à vis des cellules de cancer du sein / Despite recent therapeutic improvments, the prognosis for a patient with bone metastases (BM) remains poor, this situation prompting the research of new therapeutic strategies. Chemokines are central players in the immune response, and appear as potential targets in anti-cancer immunotherapies. We are interested to determine whether the CX3CL1 chemokine exerted pro or anti-tumor actions within the bone metastatic context. To address this issue, we developed mouse models of lung or renal cancer BM. In lung cancer BM model, our work demonstrated that CX3CL1 expression led to tumor growth inhibition. Tumors transcriptomic analysis revealed that CX3CL1: (i) impacted bone metabolism by modulating the OPG/RANKL/RANK triad (ii) decreased the expression of certain immune checkpoints, this up-regulating the anti-tumor immune response. By contrast, in renal cancer BM model, CX3CL1 expression stimulated bone tumor development and transcriptomic analysis showed that CX3CL1 (i) promoted osteolysis through an action on the OPG/RANKL/RANK triad (ii) -induced tumor development correlated with an increased expression of certain immune checkpoints, this down-regulating the anti-tumor immune response. Bisphosphonates (BPs) are targeted agents used for BM treatment. In order to reduce their side effects, we used resorbable calcium phosphate cements (CPC), which are frequently used as bone void fillers, as platform for a local delivery of BPs (alendronate, ALN). As a whole, our in vitro data demonstrated that: (i) ALN-CPC cements continuous released ALN; (ii) this ALN release was effective in inducing cytotoxic and pro-apoptotic effects in breast cancer cells

Homeostasis of hematopoietic stem cells (HSC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSC with strong implications on expansion and differentiation ex vivo as well as in vivo transplantation. Towards this aim, a cell culture analysis on a polymer microcavity carrier platform was combined with a partial least square analysis of a mechanistic model of cell proliferation. We could demonstrate the discrimination of specific autocrine and paracrine signals from soluble factors as stimulating and inhibitory effectors in hematopoietic stem and progenitor cell culture. From that we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSC in single-cell niches and advocate our analysis platform as an unprecedented option for untangling convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin.

The preclinical imaging method micro-CT (microtomography) allows the visualization and quantification of the structure of samples at a resolution of micrometers. Its' importance is increasing globally. In addition to several advantages (non-destructive, the possibility of direct 3D analysis, time efficiency, etc.), micro-CT also has some significant limitations (problematic validation of results, image artifacts, significant influence of image modifications, etc.). This thesis focuses on the application of micro-CT in the field of research and development of metallic and non-metallic materials promoting bone healing with their possible clinical applications. The first part addresses the limitations of micro-CT through several studies. A comparison of pore sizes in biomaterials utilizing scanning electron microscopy (SEM) and micro-CT was performed, and the complications of pore size evaluation were presented. SEM image analysis leads to significantly higher values than micro-CT (approximately three times), which allows for comparison of the studies using only one of these methods. Validation of micro-CT 3D analysis results based on calibration phantoms with complex structure, to date, is not possible. We therefore developed software generating phantom datasets of 3D objects with well-defined...

One of the most important factors for a successful performance of a load-bearing implant for hard tissue replacement is its mechanical compatibility with human bone. That implies that the stiffness should be close to that of a bone and the strength of the implant material must be high enough to bear the load applied under physiological conditions. The Young´s modulus of most of the commonly used biomedical alloys is larger than that of a human bone (around 100 GPa for cp Ti, 112 GPa for Ti-6Al-4V versus 10-30 GPa for cortical human bone). A stiffness reduction of Ti alloys can be achieved by two approaches: (i) selecting an alloy composition with low Young´s modulus i.e. a ß-type alloy and (ii) introducing a reasonable amount of porosity. The composition of Ti-40Nb was chosen for the present work, as it allows to stabilize a single ß-type phase with low Young´s modulus at room temperature. The samples were produced by a powder metallurgical approach. The Ti-40Nb alloy powder was obtained by ball-milling of elemental Ti and Nb powders. The influence of the milling parameters on the oxygen content in the milled powder was studied. Powders with a lowest oxygen content of 0.4 wt.-% had an almost single ß-type phase after heat treatment and quenching. Porous samples were produced by loose powder sintering, hot-pressing and sintering with NaCl as a space-holder. The influence of the different processing routes and different porosities on the mechanical properties of the alloy was studied. The samples produced by loose powder sintering had mechanical properties close to those of cortical human bone (Young´s modulus 20 GPa, compression strength 150 MPa) and the samples produced by loose sintering with space-holder materials had mechanical properties close to those of human spongy bone (Young´s modulus 0.2-2 GPa, compression strength 50 MPa). Porous Ti-40Nb samples were coated with bone-like hydroxyapatite by an electrochemical deposition method in order to improve the osseointegration of the samples with bone tissue. The experiments were carried out with samples produced by different routes and a correlation between the deposition parameters and the morphology of the hydroxyapatite needles was found.

Natural composites, in particular nacre, often combine high strength and toughness thanks to highly ordered architectures and controlled geometries of the reinforcement components. However, combining strength, toughness and resorbability in synthetic materials remains a challenge in particular in the field of bone graft substitutes. In the present study, calcium phosphate-(CaP-)based materials with designed architectures inspired from natural composite materials were achieved. CaP platelets obtained by precipitation in organic medium were first aligned in chitosan matrices by solvent casting in ambient conditions. Efficient strengthening was obtained with 15 vol-% ceramic, reaching cortical bone strength (150 MPa) and preserving good ductility (5 % deformation). In a weak magnetic field, high spatial arrangement without percolation was maintained up to 20 vol-%. With directional freezing, good alignment of the platelets could be pushed up to 50 vol-%. In parallel, in situ recrystallization of CaP blocks in hydrothermal conditions led to hierarchical structures. The strength and the work-of-fracture were enhanced (300%) thanks to a change of failure mode.:Acknowledgements v Summary vii Background vii Thesis outline viii Part I: “Brick-and-mortar” structures with discrete reinforcement components ix Part II: “Textured” structures with continuous reinforcement components x Zusammenfassung xi Hintergrund xi Doktorarbeit Gliederung xii Teil I: “Ziegelmauer-Architektur” mit diskreten Verstärkungskomponenten xiii Teil II: “ Texturierte” Strukturen mit kontinuierlichen Verstärkungskomponenten xiv Chapter 1: General introduction 1 Bone grafting 1 CaP ceramics 1 How to improve toughness of CaP ceramics? 2 Importance of structure design: bio-inspiration 2 What mechanical properties should be reached? 5 Specific aims 5 Two general approaches to reach the goal 6 Nacre-inspired “brick-and mortar” structures (Part I) 6 Textured ceramic monoliths (Part II) 6 References 7 Chapter 2: Theoretical calculations 11 Introduction 12 Theoretical tensile strength of β-TCP platelets and critical size for flaw tolerance 13 Optimal aspect ratio 15 Composite strength and stiffness 17 Limitations 19 References 19 PART I: NACRE-INSPIRED “BRICK-AND-MORTAR” STRUCTURES Chapter 3: Synthesis of sub-micrometer calcium phosphate platelets 23 Introduction 24 ii Materials and Methods 25 Precipitation method 25 Reaction parameters 25 Characterization 26 Statistical analysis of results 28 Results 28 Reproducibility of standard experiments 28 Increase of the reagent volume to increase the productivity 30 Increase of the precursors concentration to increase the productivity and modify the particles 30 Increase of titration rate to simplify the process 32 Influence of temperature on the particles 35 Effect of the pH value on the particles 37 Effect of a longer reaction time on the particle stability 40 Study of the influence of variations of the Ca/P molar ratio 42 Discussion 43 Reproducibility 43 Productivity 44 CaP crystal shape 45 Crystal purity 47 Aspect ratio 48 Critical thickness 49 Uniformity of primary particles 50 Non agglomerated 51 General points 52 Conclusions 52 References 53 Chapter 4: Kinetics study of the calcium phosphate platelets growth 57 Introduction 58 Theory 58 Materials and methods 60 Materials and sample preparation 60 Characterization methods 61 Results 62 Visual observations during manipulations 62 SEM observations 62 XRD results 66 Size measurements 68 Kinetics calculations 70 Discussion 74 Nucleation and assembly mechanism 74 Reaction kinetics 76 Control of size and aspect ratio 76 Conclusions 77 References 78 Chapter 5: Structural design of bio-inspired composites by solvent casting 81 Foreword 82 Introduction 82 Experimental section 84 iii Synthesis of resorbable ceramic platelets 84 Solvent casting to prove the reinforcement efficiency of DCP platelets 84 Magnetization of the platelets 85 Maintaining the orientation during drying of an hydrogel matrix 86 Results 87 Synthesis of resorbable ceramic platelets 87 Solvent casting to prove the reinforcement efficiency of CaP platelets 87 Magnetization of the platelets 91 Maintaining the orientation during drying of an hydrogel matrix 93 Discussion 95 Detrimental effect of β-TCP platelets in chitosan 95 Efficient reinforcement with DCP platelets up to a given volume fraction 96 Threshold value for strength improvement 97 Fitting the experimental results with theoretical equations 98 Conclusions 101 References 101 Chapter 6: Biodegradable, strong and tough nacre-inspired structures obtained by freezecasting 105 Introduction 106 Experimental section 108 Synthesis of resorbable ceramic platelets 108 Preliminary freeze-casting tests with β-TCP-based slurries 108 Determination of adequate freeze-casting parameters for hydrogels-CaP slurries 108 Integration of CaP platelets and local planar alignment 109 Attempts to globally align porosity in two directions 109 Densification and consolidation 110 Tensile testing 110 Results 111 Preliminary freeze-casting tests with β-TCP-based slurries 111 Determination of adequate freeze-casting parameters for hydrogels-CaP slurries 112 Integration of CaP platelets and local planar alignment 113 Attempts to globally align porosity in two directions 119 Densification and consolidation 121 Tensile testing 121 Discussion 122 Conclusions 124 References 125 PART II: TEXTURED CERAMIC MONOLITHS Chapter 7: Micro-texturing by recrystallization of calcium phosphate blocks in hydrothermal conditions 127 Introduction 128 Materials and Methods 130 Samples characterization 132 Results 133 Macroscopic observations 133 Microstructural changes (SEM) 133 Crystalline phase conversion (XRD) 139 iv Mechanical properties 142 Fractured surfaces 142 Discussion 145 Conclusions 150 References 150 Chapter 8: Toughening of textured calcium phosphate blocks by polymer impregnation 155 Foreword 156 Introduction 156 Materials and Methods 157 Samples preparation 157 Characterization 158 Results 158 Porosity and microstructure 158 Composition 161 Mechanical properties 161 Discussion 162 Conclusions 164 References 164 Chapter 9: Synthesis and outlook 167 Curriculum Vitae 171

Since the beginning of the 20th century plastic has been a widely used material, which has resulted in large quantities of plastic being produced in the last century. The plastics of today are mainly produced from fossil raw materials, which gives it a high climate impact. Plastic also has a long service life, which creates problems with handling after the new period when new plastic is produced at a faster rate than plastic debris can be recycled or incinerated. One possible approach is to switch from plastic from fossil sources to bioplastics, which is produced by renewable sources. This means a reduction in the environmental impact as the amount of fossil CO2 emissions from combustion of plastics would decrease. Polyhydroxyalkanoates (PHAs) are created by short volatile fatty acids (VFAs) added to bio sludge from a wastewater treatment plant at a pulp and paper mill where there is a lot of bacteria and microorganisms. Some of the bacteria in the sludge have the ability to accumulate PHA when VFA is added in excess and then be able to use it as an energy and carbon source in cases of starvation. This means that PHA produced in this way can be degraded by bacteria making it biodegradable while having similar properties as oil-based plastics. Production of PHA is currently expensive as it is often necessary to purchase VFA for production. To make it economically sustainable to replace oil-based plastics with PHA, the cost of PHA production must therefore be reduced. This can be done by using mixed bacterial cultures from, for example, industrial wastewater treatment plants and by creating their own composition of VFA through fermentation. At the paper mill at BillerudKorsnäs Gruvön there are several process streams that today are led to the water treatment plant, one of which is from PM6 (Paper Machine 6). By fermenting that stream, VFA can be formed with a composition of acetic acid, propionic acid, and butyric acid. The current may thus be suitable to use as a substrate in PHA production. Another way to produce VFA is to ferment the residual flow from hydrothermal carbonation (HTC) of bio sludge. According to (Samorì et al., 2019), acetic acid, butyric acid and valeric acid are formed, which means that even this stream may be suitable as a substrate for PHA production. The purpose of this thesis is to investigate the effects of the composition of VFA on the production of PHA from forest industrial bio sludge. The study covers two different cases, Case PM6 and Case HTC, where two different types of composition of VFA are added to paper sludge from the paper mill. The experiments were performed in cylindrical tanks on three occasions where the first two experiments had a volume of 30 liters and the last experiment a volume of 10 liters. On the first occasion, the maximum accumulation rate in the growth phase was studied. In the second instance, a high accumulation rate was sought, but also survival after the growth phase. The first two trials were batch trials where the sludge was dosed until saturation was achieved. On the third occasion, the possibility of carrying out PHA production with a continuous sludge exchange was studied. The experiments were analyzed by FTIR which provided information on the absorbance of the sludge which shows how the PHA concentration increased during the course of the experiments. Extractions were then performed to obtain the concentration of PHA that eventually accumulated in the sludge. The results show that biomass from BillerudKorsnäs Gruvön's mills accumulated PHA faster with VFA composition from fermented PM6 effluent compared to VFA composition from fermented HTC condensate. Calculations made with input from the experiments indicate that it is possible to produce a larger amount of PHA per year with Case PM6. The conclusion is therefore that Case PM6 is preferable if as large a PHA production as possible wants to be achieved. Case HTC is instead preferred if reduced PHA production can be tolerated in favor of biocarbon production.

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Biomaterials are designed to improve impaired healing of injured tissue. To accomplish better cell integration, we suggest to coat biomaterial surfaces with bio-functional proteins. Here, a mussel-derived surface-binding peptide is used and coupled to CXCL12 (stromal cell-derived factor 1α), a chemokine that activates CXCR4 and consequently recruits tissue-specific stem and progenitor cells. CXCL12 variants with either non-releasable or protease-mediated-release properties were designed and compared. Whereas CXCL12 was stabilized at the N-terminus for protease resistance, a C-terminal linker was designed that allowed for specific cleavage-mediated release by matrix metalloproteinase 9 and 2, since both enzymes are frequently found in wound fluid. These surface adhesive CXCL12 derivatives were produced by expressed protein ligation. Functionality of the modified chemokines was assessed by inositol phosphate accumulation and cell migration assays. Increased migration of keratinocytes and primary mesenchymal stem cells was demonstrated. Immobilization and release were studied for bioresorbable PCL-co-LC scaffolds, and accelerated wound closure was demonstrated in an ex vivo wound healing assay on porcine skin grafts. After 24 h, a significantly improved CXCL12-specific growth stimulation of the epithelial tips was already observed. The presented data display a successful application of protein-coated biomaterials for skin regeneration.