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Ce travail a pour objet de déterminer les conditions optimales de production de substituts allogéniques capables de combler les lésions cartilagineuses dans le cadre du traitement de l’arthrose. Il s’oriente particulièrement sur la composante cellulaire de ces substituts. L’usage de cellules souches mésenchymateuses issues de cordons ombilicaux (CSM-GW) implique de déterminer quels facteurs obstétricaux, liés à l’environnement direct et indirect des CSM-GW, peuvent influencer leur prolifération ainsi que leur différenciation chondrocytaire. Dans une première partie de ce travail, trois types de facteurs ont été étudiés : les facteurs liés à l’enfant donneur, au déroulement de l’accouchement et de la délivrance, à la grossesse et à la mère. Nos données montrent que les CSM-GW ont des capacités prolifératives améliorées lorsque l’accouchement s’est déroulé à terme et sans complication, avec utilisation de Syntocinon® pendant le travail. Sur la base de ces résultats, nous avons utilisé les CSM-GW les plus efficaces dans le cadre de l’ingénierie du cartilage. Il a ensuite été essentiel d’élucider le profil d’action des CSM-GW dans un contexte allogénique. Le deuxième temps de ce travail a donc consisté à chercher le profil de stimulation le plus performant, au regard de la viabilité des cellules et de l’évolution de la sécrétion des facteurs solubles responsables des propriétés immunomodulatrices des CSM-GW au cours de la différenciation chondrocytaire. Nous avons alors mimé, in vitro et en biomatériaux d’Alginate/Acide hyaluronique (Alg/HA) une telle situation en stimulant les CSM-GW avec différentes doses d’IFN-γ et de TNF-α. Selon nos résultats, la stimulation par IFN-γ et TNF-α sur les CSM-GW en biomatériaux d’Alg/HA est plus efficace lorsque ces deux cytokines sont utilisées conjointement et n’est pas délétère pour la viabilité cellulaire aux concentrations respectives de 20 et 30 ng/mL. Cette double stimulation induit une augmentation de la sécrétion d’IL-6 et de PGE-2 par les CSM-GW, ne modifie pas leur sécrétion de TGF-β, et diminue la sécrétion de VEGF. Nous avons confirmé ces données lors d’une mise en situation fonctionnelle : des cocultures avec des cellules mononucléées de sang périphérique (PBMC) de donneurs sains nous ont permis d’évaluer la réponse des CSM-GW lors d’une situation allogénique. Ces mises en situations allogéniques ont été étudiées à différents temps afin d’évaluer les propriétés immunologiques des CSM-GW au cours du temps passé en biomatériaux. Nos résultats montrent que les CSM-GW peuvent exprimer des molécules HLA-G ainsi qu’IDO, mais ces expressions sont limitées en biomatériaux d’Alg//HA. Les CSM-GW en biomatériaux d’Alg/HA en situation allogénique ne sont pas immunogènes, quel que soit le temps de différenciation. En revanche, leurs capacités immunomodulatrices décroissent au cours du temps et sont plus fortes à J0 et J3 de la différenciation chondrocytaire, ce qui oriente vers une utilisation précoce de ces cellules. Les conclusions de ce travail permettent de (i) sélectionner les cordons idoines à l’ingénierie cellulaire et l’ingénierie du cartilage, (ii) définir les conditions permettant de mimer une situation allogénique in vitro, (iii) connaitre les propriétés immunomodulatrices des CSM-GW au cours de la culture en biomatériaux d’Alg/HA, y compris en situation allogénique / The purpose of this work is to determine the optimal conditions for allogeneic substitutes production, adapted to filling the cartilaginous lesions in osteoarthritis treatment. It focuses on the cellular component of these substitutes. The use of mesenchymal stem cells from umbilical cords (WJ-MSC) involves determining which factors, related to direct and indirect environment of the WJ-MSC, can influence their proliferation and chondrogenic differentiation. In a first part of our work, three types of factors were studied: related to the donor child, the course of labor and delivery, pregnancy and the mother. Our results show that WJ-MSC have enhanced proliferative capacities when coming from full-term birth and without complications, with the use of Syntocinon® during labor. On this basis, we used the most effective WJ-MSC for cartilage engineering. It was then essential to elucidate their action profile in allogeneic context. We stimulated WJ-MSC embedded in Alginate/Hyaluronic Acid (Alg/HA) scaffolds with different concentrations of IFN-γ and TNF-α in order to determine the most effective stimulation profile, with regard to viability of the cells and evolution of immunomodulatory soluble factors secretion. According to our results, the stimulation by IFN-γ and TNF-α on WJ-MSC in Alg/HA scaffolds is more effective when these two cytokines are used together and is not deleterious for cell viability at the concentrations of 20 and 30 ng/mL, respectively. This double stimulation induces an increase in the secretion of IL-6 and PGE-2 by the WJ-MSC, a decrease in the secretion of VEGF and does not modify the secretion of TGF-β. We confirmed these data during a functional study: cocultures with peripheral blood mononuclear cells (PBMC) from healthy donors allowed us to evaluate the response of WJ-MSC in an allogeneic situation. These allogeneic situations have been studied at different times to evaluate the immunological properties of WJ-MSC during the time of chondrogenic differentiation. Our results show that WJ-MSC can express HLA-G molecules as well as IDO, but these expressions are limited in Alg/HA biomaterials. Finally, the WJ-MSC in Alg/HA biomaterials in allogeneic conditions are not immunogenic, regardless of the time of differentiation. On the other hand, their immunomodulatory capacities decrease over time and are stronger at day 0 and day 3 of chondrogenic differentiation, which leads to an early use of these cells. Finally, this work allows us to (i) select the umbilical cords suitable for cellular and cartilage engineering, (ii) define the conditions mimicking in vitro an allogeneic situation, (iii) elucidate the immunomodulatory properties of WJ-MSC during Alg/HA biomaterials chondrogenic differentiation, including allogeneic situations

Magister Scientiae - MSc (Medical BioSciences) / In the past three decades there has been a huge increase in the use of herbal medicine globally. The active principles of these herbal medicines are mostly unknown with supportive evidence for safety and efficacy very rare. SeptilinTM is a phytopharmaceutical formulation which is recommended for the treatment and management of various infections. It has been claimed to have immunomodulatory actions that potentiates the body's immune response. The immunomodulatory activity of SeptilinTM has not been well investigated via appropriate in vitro models. Therefore this study was undertaken to investigate the in vitro effects of SeptilinTM on biomarkers of specific immune pathways by using WBC. Stimulated and unstimulated WBC were incubated with the product. Enzyme linked immunosorbent assays were used to screen for IL-6, IL-10, and IFN? as biomarkers for inflammation, humoral immunity, and cell mediated immunity, respectively. Results show that the presence of SeptilinTM in LPS stimulated WBC has no effect on the release of IL-6 and IFN? production but stimulated IL-10 production. SeptilinTM in unstimulated WBC has no effect on the release of IL-10 and IFN? production but stimulatory effects on IL-6 production.

The aim of the thesis was development of an innovative treatment of bone defects. Human multipotent mesenchymal stromal cells (MSC) play a crucial role in bone healing. Clinical applications of MSC require large amount of cells, which could be obtained by autologous expansion of MSC harvested from bone marrow. As a first step, the standard protocol of MSC expansion based on αMEM medium and fetal bovine serum (FBS) was used. Experiments replacing FBS by pooled human serum (HS) in the culture medium concluded in patenting of a new MSC cultivation protocol (EU 1999250, CR 301141). This one-step cultivation protocol and xenogeneic protein-free cultivation medium is based on CellGro® for Hematopoietic Cells' Medium, HS, human recombinant growth factors, dexamethasone, insulin and ascorbic acid. The preclinical in vitro and in vivo experiments with MSC from both expansion protocols were carried out. Fibrillar polylactic scaffolds were seeded with MSC, cultured, differentiated and implanted in immunodeficient mice (NOD/LtSz-Rag1-). Bone-like mineralized tissue containing vessels was observed. The MSC cultured according to patented method were classified as Advanced-therapy Medicinal Product and has to fulfil the European Medicines Agency regulations to enter the clinical trials. Nevertheless the use of MSC seems...

Notre équipe a optimisé la formulation de dispositifs médicaux implantables pour l’ingénierie tissulaire osseuse et cartilagineuse. A ces fins, nous nous sommes basés sur des implants nanostructurés d’origine naturelle ou synthétique conçus au sein du laboratoire par la méthode d’électrospinning, pour imiter la matrice extracellulaire du compartiment osseux, et un hydrogel composé d’alginate et d’acide hyaluronique imitant la composition du compartiment cartilagineux. Dans une première partie de mon travail, pour la régénération osseuse, nous avons optimisé la formulation d’un implant nanostructuré à base de chitosane pour une accélération de cette régénération. Ceci a été possible en rendant actif ce dispositif médical implantable par incorporation de nanoparticules de silice, conférant à la construction nanocomposite des propriétés mécaniques accrues, et une excellente biocompatibilité avec le tissu hôte. Une autre étude pour la même visée a permis d’élaborer une nouvelle stratégie d’ensemencement de dispositif implantable synthétique et nanostructuré par des microtissus cellulaires, remplaçant un ensemencement de cellules isolées et permettant des performances de minéralisation accrues à l’intérieur de l’implant. Dans un deuxième temps, pour la régénération de l’unité ostéoarticulaire, nous avons proposé deux implants bi-compartimentés et hybrides comportant des microtissus de cellules souches mésenchymateuses. Ces implants sont composés d’un hydrogel contenant les cellules souches permettant la régénération du cartilage, et d’une membrane collagénique naturelle (Bio-Gide®) ou synthétique (membrane de polycaprolactone), dotée de nanoréservoirs (technologie brevetée par le laboratoire) de facteur de croissance ostéogénique (BMP-7) pour une régénération du socle osseux (os sous-chondral) de l’unité os-cartilage. La troisième partie de mon travail a concerné la vascularisation des implants osseux et particulièrement l’accélération du recrutement vasculaire. Dans ce cadre plus vasculaire, nous avons proposé une stratégie qui vise à doter un implant synthétique nanostructuré de facteur de croissance angiogénique (VEGF), puis à lui appliquer un ensemencement séquentiel de cellules mésenchymateuses adultes « ostéoblastes humains» et de cellules endothéliales humaines (HUVECs). Cette stratégie a permis un recrutement et une hiérarchisation accrue des cellules endothéliales dans l’implant. En conclusion, l’optimisation des implants développés au laboratoire permettra sans nul doute de proposer dans un futur proche de nouveaux dispositifs médicaux implantables (DMI) thérapeutique combinés de type DMI-MTI (Médicaments de Thérapie Innovante) pour l’ingénierie tissulaire osseuse et cartilagineuse en particulier en médecine régénérative ostéo-articulaire. / Our team optimized the formulation of implantable medical devices for bone and cartilage tissue engineering. To that end, we based our work on nanostructured implants, either natural or synthetic, made in the laboratory by electrospinning process, to mimic bone extracellular matrix, and hydrogel of alginate/hyaluronic acid to mimic cartilage extracellular matrix. First, concerning bone regeneration, we optimized the formulation of a nanostructured scaffold composed of natural chitosan to enhance bone regeneration. This was made possible by doping this implantable medical device with silica nanoparticles, offering this nanocomposite better mechanical properties, and excellent biocompatibility with host tissue. Another study with the same aim allowed elaborating a new cell seeding strategy, to seed these implantable medical devices with cell microtissues instead of single cells, offering higher mineralisation efficiencies within the implant. Consequently, for the regeneration of the osteochondral unit, we proposed two compartmented and hybrid implants comprising mesenchymal stem cells microtissues. Those implants are made of a hydrogel containing the stem cells, allowing the regeneration of cartilage, and a membrane, either natural (collagenic Bio-Gide®) or synthetic (electrospun polycaprolactone) equipped with nanoreservoirs (technology patented by the laboratory) of osteogenic growth factor (BMP-7) for the regeneration of osseous stand (the subchondral bone) of the bone-cartilage unit. Finally, to study the improvement in vascular recruitment, we proposed a new strategy combining the modification of an implantable device with angiogenic growth factor (VEGF), prior to its sequential seeding with mesenchymal cells “human osteoblasts” and human endothelial cells (HUVECs). This strategy allowed higher recruitment and structuration of endothelial cells within the implant. To conclude, the implant optimisation strategies developed in the laboratory will certainly allow proposing in the near future new combined Advanced Therapy Medicinal Products (ATMPs) and Implantable Medical Device for bone and cartilage regeneration, in particular in the field of osteoarticular regenerative nanomedicine.

This diploma thesis deals with current pharmacotherapeutic care with a focus on activities that nurses take part in. The aim was to collect and evaluate the views and experiences of selected nurses regarding their role in pharmacotherapy team, particularly what activities they perform in such team and how their pharmacotherapy teams work in general. SWOT analysis was then used firstly to evaluate the current role of nurses and secondly to suggest a way of implementation of possible extension of nurses' competencies in pharmacotherapy. In order to collect the required data, semi-structured interviews were conducted. These were recorded, so they could be easily processed afterwards. During transcription of the interviews into written text, all the collected data was briefly analysed. The data was further analysed using the open coding method, when three main categories with specific subcategories and subcodes were identified. All the selected men and women, which were asked for cooperation, are people working as a nurse without supervision and mentoring. Based on their career history all of them were expected to have a more specific experience with the issue. The respondents were mainly nurses with long-term working experience, often working in middle or senior management. When selecting the future...

The aim of the thesis was development of an innovative treatment of bone defects. Human multipotent mesenchymal stromal cells (MSC) play a crucial role in bone healing. Clinical applications of MSC require large amount of cells, which could be obtained by autologous expansion of MSC harvested from bone marrow. As a first step, the standard protocol of MSC expansion based on αMEM medium and fetal bovine serum (FBS) was used. Experiments replacing FBS by pooled human serum (HS) in the culture medium concluded in patenting of a new MSC cultivation protocol (EU 1999250, CR 301141). This one-step cultivation protocol and xenogeneic protein-free cultivation medium is based on CellGro® for Hematopoietic Cells' Medium, HS, human recombinant growth factors, dexamethasone, insulin and ascorbic acid. The preclinical in vitro and in vivo experiments with MSC from both expansion protocols were carried out. Fibrillar polylactic scaffolds were seeded with MSC, cultured, differentiated and implanted in immunodeficient mice (NOD/LtSz-Rag1-). Bone-like mineralized tissue containing vessels was observed. The MSC cultured according to patented method were classified as Advanced-therapy Medicinal Product and has to fulfil the European Medicines Agency regulations to enter the clinical trials. Nevertheless the use of MSC seems...

ATMP (Advanced Therapy Medicinal Products) is a new type of treatment with the potential to cure otherwise severe and even deadly diseases, for which there are only inhibitive medicine available. This means that the healthcare sector now have access to treatments that greatly increases the number of QALY:s received. However, the problem with the implementation of ATMP lies within the disparity between its large upfront costs and the return, which is spread over a long time-period. There is also the problem with lack of information, causing further risks when investing in ATMP. Seeing as the treatment is a “one-off” where the results are uncertain, and the costs are high and lumped together at the beginning. By constructing a laboratory setting we analyze the health evaluating outcome when changing endogenous variables and modelling scenarios from real world estimates, forming a hypothetical base case which represents the difficulty decision makers face when evaluating treatments with ATMP- characteristics. Furthermore, we find that there is a prominent cost difference between the two alternatives as well as an enhanced sensitivity to the methodologically execution. To mitigate the risk of these types of long-term investments we present different types of strategies, which are built upon financial theory by the utilization of options and swaps. We also suggest using these payment schemes based upon our results, this can open opportunities to elude the decision uncertainty present for these types of long-term investments within the public healthcare sector. Making use of real options and venture capitalist structures we present a variety of solutions that might be suitable for different types of scenarios and ATMP:s. These different types of payment solutions and risk mitigating strategies could potentially also be used in combination with each other depending on the availability of a counterparty and the duration of the contract. In conclusion, we find that financial theory can mediate decisionmakers in situations that suffer from ATMP characteristics. We find financial solutions that can price long-term uncertainty and alleviate irreversibility from decisionmakers. Relying on already preferred solutions in the health sector i.e., negotiated contracts and outcome-based payment structures, financial theory has the possibility to use public funds more effectively with mitigated uncertainty. / ATMP (Avancerade terapiläkemedel) är en ny typ av behandling som har potentialen att bota i andra fall allvarliga och även dödliga sjukdomar, för vilka det enbart finns lindrande åtgärder eller inga alternativa behandlingar alls. Detta betyder att sjukvården numer har tillgång till behandlingar som ökar antalet erhållna QALY:s avsevärt. Problematiken vid implementeringen av ATMP ligger däremot i de stora engångskostnaderna som tillkommer direkt och de hälsoeffekter som de ger, vilka återfinns på lång sikt. Det finns även en problematik i informationsbristen, vilket skapar risker vid investeringar i ATMP. Anledningen till detta är att behandlingen är en engångsbehandling med osäkra resultat och höga kostnader. Genom att konstruera en analysmodell skapar vi ett basscenario utifrån vilket vi kan ändrade variabler för att generera olika utfall som ska representera underlaget till vilket beslutfattare ska agera. Vid 1000 iterationer kan vi visa hur ATMP är överlägset en konventionell behandling för t.ex. akut lymfatisk leukemi när det kommer till att generera QALY:s. Vidare finner vi att det finns en framträdande kostnadsskillnad mellan de två alternativen samt en ökad känslighet för metodologiskt utförande. För att främja beslutsprocessen och hantera risken vid den här typen av långsiktiga investeringen lägger vi fram olika typer av strategier som är rotade i finansiell teori genom nyttjandet av optioner och swappar. Vi föreslår betalningslösningar som är baserade på de resultat vi fick och som har möjligheten att minska beslutosäkerheten som uppstår vid den här typen av långsiktiga investeringar inom sjukvården. Genom att använda realoptioner och riskkapitaliststrukturer ger vi en bred mängd lösningar som kan vara lämpliga för olika typer av scenarion och avancerade terapiläkemedel. Dessa olika typer av betalningslösningar och riskhanteringsstrategier kan potentiellt även användas i kombination med varandra, beroende på tillgången till en motpart och längden på kontraktet. Sammanfattningsvis finner vi att finansiell teori kan hjälpa beslutsfattare i situationer som lider av ATMP karakteristik. Vi föreslår finansiella lösningar som kan hjälpa till med att prissätta osäkerhet på lång sikt och minska irriversibiliteten från beslutsfattaren. Vi använder väletablerade metoder och föredragna betalningslösningar, det vill säga förhandlade kontrakt och evidensbaserade betalningsstrukturer, tillsammans med finansiell teori för att konstruera betalningslösningar som använder offentliga medel mer effektivt och ger lägre osäkerhet.

(in English): The subject of my dissertation is the decision of the subjects to participate in the early phase of clinical trial. The aim of this disseration is to detect and understand the ethical aspects of decision making of subjects with oncological diseases to participate in the early phase of clinical trial. In the theoretical part of my dissertation, I define the field of clinical research, describe the phases of clinical research and ethical codes important for clinical research. The current ethical dilemmas related to the participation of subjects in the early phase of clinical trial, which are being discussed by the professional public, are presented. The research part contains the results of interviews with seventeen respondents whose content correlates with the aim of the research part and it is to understand the decision- making of subjects to participate in the early phase of clinical trial in terms of subjectively constructed realities, which are different for individual respondents. The results of the research show that fourteen respondents identified one of the most important aspects of the decision-making to participate in the early phase of clinical trial of the recommendation of principal investigator and trust in a physician. Twelve respondents stated the most frequent reason...

(in English): The subject of my dissertation is the decision of the subjects to participate in the early phase of clinical trial. The aim of this disseration is to detect and understand the ethical aspects of decision making of subjects with oncological diseases to participate in the early phase of clinical trial. In the theoretical part of my dissertation, I define the field of clinical research, describe the phases of clinical research and ethical codes important for clinical research. The current ethical dilemmas related to the participation of subjects in the early phase of clinical trial, which are being discussed by the professional public, are presented. The research part contains the results of interviews with seventeen respondents whose content correlates with the aim of the research part and it is to understand the decision- making of subjects to participate in the early phase of clinical trial in terms of subjectively constructed realities, which are different for individual respondents. The results of the research show that fourteen respondents identified one of the most important aspects of the decision-making to participate in the early phase of clinical trial of the recommendation of principal investigator and trust in a physician. Twelve respondents stated the most frequent reason...