Des résultats récents ont montré les premières preuves d’un effet multivalent puissant des iminosucres sur l’inhibition des glycosidases, avec des gains d’affinité allant jusqu’à 10000. Afin de comprendre les différentes caractéristiques de cet « effet de cluster » et d’en poursuivre l’optimisation, de nouvelles charpentes doivent être conçues. Le premier axe de recherche a donc consisté en la mise au point d’un ensemble de techniques d’ingénierie « click » pour la synthèse de systèmes multivalents, avec le développement d’une stratégie Sweet LEGO®. Cette approche permettrait un accès simple à une grande variété de néocyclodextrines préfonctionnalisées. Le second axe a consisté en une étude de relation structure-activité autour de charpentes moléculaires polyalcynes utilisées pour préparer de nouveaux clusters par chimie « click ». Une partie des clusters a été préparée en utilisant de nouvelles charpentes cyclopeptoïdes. Ils ont permis de poursuivre l’optimisation de l’effet multivalent des iminosucres sur l’inhibition de glycosidases. En particulier, un composé portant 30 ligands a montré le meilleur effet multivalent connu sur une enzyme modèle. / Recent reports have demonstrated the first pieces of evidence of a strong multivalent effect in glycosidase inhibition by iminosugars, with affinity enhancements close to 10000. In order to understand the different parameters of this “cluster effect” and to continue its optimization, new scaffolds must be designed. The first research topic was thus to develop a set of « Click » Chemistry engineering techniques for the synthesis of multivalent systems, with the development of a Sweet LEGO® strategy. In the end, it would allow an easy access to a broad range of prefunctionalized neocyclodextrins. The second research topic consisted in a structure-activity relationship study by varying the molecular polyalkyne scaffold used for the preparation of new clusters by way of « Click » Chemistry. They allowed to investigate the specific features of the iminosugar cluster effect in the inhibition of glycosidases. In particular, a compound of unprecedented valency bearing 30 iminosugar units demonstrated an unprecedented dramatic affinity enhancement for the inhibition of a model enzyme (Jack bean alpha-mannosidase).

Abstract Bone tissue engineering has become a rapidly expanding research area because it offers a promising new approach for bone repair and regeneration. Compared to traditional autograft and allograft procedures, bone tissue engineering techniques based on the use of scaffolding materials in combination with autogenous stem cells can eliminate problems of donor site morbidity associated with the harvest of bone tissue, and its short supply. Clearly, the choices of material as well as a scaffold design that enhance bone regeneration are major challenges in the tissue engineering approach. Fibers in the micro-range in combination with textile-based technologies are consider as potential routes for the production of complex scaffolds since they can be used to generate a wide range of morphological structures and geometrically varied structures with high precision. Therefore in this thesis the specific objects were to: (i) develop a biocompatible composite fiber from poly(lactic acid) (PLA) and hydroxyapatite (HA) by melt spinning, (ii) design a 3D textile scaffold utilizing weaving and (iii) evaluate the scaffolds’ performance as a bone substitute material in vitro. In the present study PLA/HA composite fibers were successfully produced, and found to possess sufficient mechanical strength even at high loading concentrations (i.e. 20wt %), to be useful in a textile process. In addition, the material was shown to be biocompatible and the presence of HA in the PLA composite significantly enhanced the initial cell attachment. In a 3D woven scaffold, bone marrow derived human mesenchymal stem cells (hMSCs) differentiated into osteoblasts and mineralized bone formation in vitro was observed through-the-thickness of the scaffold. Taken together, these results indicate the potential feasibility of PLA/HA composite fiber in a 3D woven scaffold for use as a bone substitute material in tissue engineering applications. / Tiivistelmä Luupuutosten korvaaminen kudosteknologisesti on kehittynyt nopeasti ja tutkimustulokset tarjoavat lupaavia mahdollisuuksia tuottaa uutta luuta luupuutosalueelle. Perinteisiin potilaan omasta luusta tehtyihin luusiirteisiin ja pankkiluusiirteisiin verrattuna potilaan omat kantasolut voivat vähentää ongelmia, joita ovat siirremateriaalin rajallinen saatavuus ja vieraan kudoksen aiheuttamat reaktiot. On tärkeä etsiä hyviä materiaaleja, joista voidaan valmistaa sellaisia kolmiulotteisia (3D) rakenteita, joilla tehostetaan luun paranemista ja uuden luun muodostumista. Kutomalla tuotetut tukirakenteet mahdollistavat kantasolusiirteille kolmiulotteisuuden, jota voidaan säädellä monipuolisesti ja tarkasti. Tämän väitöstutkimuksen tarkoituksena oli: (i) kehittää bioyhteensopiva kuitu maitohappopolymeeristä poly lactic acid (PLA) ja hydroksiapatiitista (HA) kuituekstruusiolla, (ii) suunnitella ja kutoa tästä kuidusta 3D tekstiilirakenne, ja (iii) tutkia sen toimivuus ja ominaisuudet luunmuodostusta tukevana materiaalina soluviljelyolosuhteissa. Tämä tutkimus osoittaa, että PLA kuitua voidaan seostaa hydroksiapatiitilla, ja PLA/HA kuidut ovat mekaanisesti kestäviä sisältäessään jopa 20 painoprosenttia hydroksiapatiittia. Siten kuidut ovat tekstiilin valmistuksessa käyttökelpoisia. Lisäksi materiaali osoittautui bioyhteensopivaksi, ja hydroksiapatiitti paransi solujen tarttumista PLA kuituun viljelyn alkuvaiheessa. Ihmisen luuytimestä peräisin olevat sidekudoksen kantasolut (hMSCs) erilaistuivat soluviljelmässä luuta muodostaviksi soluiksi eli osteoblasteiksi, ja tuottivat mineralisoitunutta luun väliainetta kautta koko kudotun tukirakenteen. Johtopäätöksenä on, että PLA/HA yhdistelmäkuitua voidaan kutoa kolmiulotteiseksi tukirakenteeksi, ja sitä on mahdollista käyttää apuna korvattaessa luupuutoksia kudosteknologian keinoin.

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.

Ice-templating, znm tak© jako freeze-casting, je relativnÄ jednoduchou, levnou a velmi univerzln­ technikou pro vrobu por©zn­ch keramickch struktur s ­zenou mikrostrukturou. Takto pipraven© keramick© struktury jsou pouity pro vrobu hybridn­ch keramickch kompozit, nebo jako biokeramick© scaffoldy. Hybridn­ keramick© kompozitn­ materily jsou zaloeny na napodobovn­ p­rodn­ch/ biologickch materil. Hlavn­m c­lem je napodobit v p­rodÄ se vyskytuj­c­ zhouevnauj­c­ mechanismy t­m, e por©zn­ keramick© struktury jsou po slinut­ naputÄny polymern­mi materily. Hlavn­m probl©mem pi vrobÄ por©zn­ch keramickch vzork s velkmi rozmÄry, pomoc­ metody ice-templating, je dosaen­ ­zen©ho rstu ledovch krystal v cel©m objemu vzorku. Aby tedy bylo mon© z­skat velk© keramick© vzorky s dobe definovanou lamelrn­ strukturou je teba proces ice-templatingu velmi pesnÄ kontrolovat. Biologick aktivita biokeramickch materil zvis­ na kombinaci fyzikln­ch a chemickch charakteristik, kter© silnÄ souvisej­ s jejich mikrostrukturou. Porozita scaffold mus­ bt vzjemnÄ propojen a velikost­ pr dostateÄnÄ velk pro spÄn rst kostn­ tknÄ v cel©m objemu implanttu. Prezentovan disertaÄn­ prce je zamÄena na problematiku zvÄtovn­ rozmÄr keramickch vzork pipravench pomoc­ metody ice-templating, vytvoen­ v­cerovov© porozity uvnit vzork a vrobu hybridn­ch keramickch kompozit pro balistickou ochranu. Keramick© suspenze pro ice-templating byly spÄnÄ pipraveny z rznch prk (zejm©na hydroxyapatitu a oxidu hlinit©ho s rznm plnÄn­m keramick©ho prku od 7,5 obj.% do 45 obj.%. Byl tak© studovn vliv aditiv na utven­ lamelrn­ drsnosti a mezilamelrn­ch pemostÄn­. V souÄasnosti je zkoumn dopad tÄchto strukturn­ch prvk na vsledn© mechanick© vlastnosti. Hybridn­ kompozity oxid hlinit/polymer byly spÄnÄ navreny a pipraveny z destiÄek z oxidu hlinit©ho pipravench metodou ice-templating s d©lkou lamel a 70 mm a rznch polymern­ch pryskyic. Byla testovny mechanick© vlastnosti hybridn­ch kompozit oxid hlinit/polymer a vsledky ukzaly, e ice-templating je robustn­ metodou pro vrobu hybridn­ch kompozit keramika-polymer s dobrm pomÄrem pevnost/hustota. Avak balistick© testy hybridn­ch kompozit oxid hlinit/polymer odhalily, e vÄtina kompozit vytvoench v rmci t©to prce nebyla schopna ÄinnÄ zastavit stely s prbojnm jdrem. Ukzalo se, e kombinace procesu ice-templating a nep­m©ho 3D tisku umouje vrobu biokeramickch scaffold pro kostn­ nhrady z hydroxyapatitu s v­cerovovou porozitou, co by se mohlo ukzat jako prospÄn© pro vvoj bioaktivn­ch vysoce por©zn­ch scaffold se zvenou biologickou aktivitou. Ice-templating tak© vznamnÄ ovlivnil zmÄnu fzov©ho sloen­ bÄhem slinovn­ hydroxyapatitovch vzork.

Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...

Experimental models of a transfer of stem cells for therapeutic purposes Abstract Stem cell therapy currently represents a standard procedure of treating a wide variety of hereditary diseases and serious injuries. Development of the most suitable way of transfer of stem cells into the patient body remains very important question concerning this type of therapy. In our experiments we used nanofiber scaffolds for stem cell cultivation and their subsequent transfer. These nanofibers were prepared by the original needleless electrospun NanospiderTM technology. Allogeneic cornea or skin graft were transplanted from B6 mice to BALB/c mice. The grafts were covered by a nanofibrous scaffold with cultivated stem cells. Stem cells were stained by an imunofluorescent dye to enable us to monitore their migration from nanofibers into tissues and consequent distribution in the body and characterize changes of this distribution in the time. The methods of ELISA and PCR were used to confirm that mesenchymal stem cells support the production of antiinflammatory cytokines IL-4 and IL-10 and contribute to inhibition of production of proinflammatory cytokines IL-1, IFNγ and inducible nitric oxide synthase. We confirmed an important beneficial role of nanofiber scaffolds in transplantation of mesenchymal stem cells. Nanofiber...

Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...

Over 600,000 bone-grafting operations are performed each year in the United States. The majority of the bone used for these surgeries comes from autografts that are limited in quantity or allografts with high failure rates. Current synthetic bone grafting materials have poor mechanical properties, handling characteristics, and bioactivity. The goal of this dissertation was to develop a clinically translatable bone tissue engineering scaffold with improved handling characteristics, bioactivity, and smart delivery modalities. We hypothesized that this could be achieved through the rational selection of Food and Drug Administration (FDA) approved materials that blend favorably with hydroxyapatite (HA), the principle mineral component in bone. This dissertation describes the development of smart bone tissue engineering scaffolds composed of the biodegradable amphiphilic polymer poly(D,L-lactic acid-co-ethylene glycol-co- D,L-lactic acid) (PELA) and HA. Electrospun nanofibrous HA-PELA scaffolds exhibited improved handling characteristics and bioactivity over conventional HApoly( D,L-lactic acid) composites. Electrospun HA-PELA was hydrophilic, elastic, stiffened upon hydration, and supported the attachment and osteogenic differentiation of rat bone marrow stromal cells (MSCs). These in vitro properties translated into robust bone formation in vivo using a critical-size femoral defect model in rats. Spiral-wrapped HA-PELA scaffolds, loaded with MSCs or a lowdose of recombinant human bone morphogenetic protein-2, templated bone formation along the defect. As an alternate approach, PELA and HA-PELA were viii rapid prototyped into three-dimensional (3-D) macroporous scaffolds using a consumer-grade 3-D printer. These 3-D scaffolds have differential cell adhesion characteristics, swell and stiffen upon hydration, and exhibit hydration-induced self-fixation in a simulated confined defect. HA-PELA also exhibits thermal shape memory behavior, enabling the minimally invasive delivery and rapid (>3 sec) shape recovery of 3-D scaffolds at physiologically safe temperatures (~ 50ºC). Overall, this dissertation demonstrates how the rational selection of FDA approved materials with synergistic interactions results in smart biomaterials with high potential for clinical translation.

Year 2019, football, rugby, netball and skiing had most occurring ACL injuries, listed by United Kingdom National Ligament Report (NLR). The standard procedure treatment of complete laceration of the ACL, is performed by tissue autograft implantation designed from a patellar tendon, for replacement of damaged tissue using orthopaedic surgery. The aim of this thesis is to design and fabricate an ACL graft, attempting to mimic the natural ACL, for the purpose of tissue reconstruction. The desired graft analogues exhibited properties imitating native connective tissue, reducing pain through drug delivery with great biocompatibility and enhance suture mechanical strength. Various biomaterials were implemented into this study, utilising strategies; polymer solution fabrication, electrospinning, hydrogel synthesis, mechanical braiding and graft assembly to fabricate an ACL graft. The polymeric material poly (E- caprolactone) (PCL) was researched, utilising its ability to fabricate scaffolds. Results showed, three analogue ACL grafts (Braided PCL-BP, Braided PCL + Hydrogel-BPH & Braided PCL + Sutures-BPS) created utilising the properties of braiding, hydrogels and sutures, ultimately improving the versatility of electrospinning for tissue engineering and reconstruction. Graft analogues were tested and compared against patellar tendons producing similar tensile properties. Poly vinyl alcohol (PVA) hydrogels successfully held ibuprofen, revealing drug delivery characteristics, polyester threads improved mechanical properties of electrospun grafts and dry degradation showed that PCL did not lose significant mass over two months. Conclusion, tensile strength of patella tendon was 395x, 790x & 56x of analogue grafts (BP, BPH & BPS) respectively, having potential for improvement of tensile parameters for ligament reconstruction.

In this thesis, micro computed tomography (μ−CT) scans of a bio-glass scaffold produced by the robocasting technique was used to create finite element method (FEM) models with the purpose of determining its mechanical strength. Prior to this, a Matlab script was used to create several simplified geometries of the scaffold in an effort to determine the importance of scaffold design parameters (such as the fiber compenetration between two adjacent printing planes) on the strength of the scaffold. Furthermore, to assess the influence of micro-structural defects such as voids and micro-cracks that are intrinsic to the robocasting manufacturing process, the total number of voids and their respective volume was calculated using the μ-CT scan imagery and fitted to a statistical distribution. The distribution of voids was then used to create several scaffold models in Matlab with either spherical or ellipsoidal voids present. In the final two models, one scaled-down and one scaled-up FEM based on μ-CT scans were investigated.To model the crack initiation, propagation and final failure, the phase-field method was used. The method was implemented by the use of a publicly available Fortran user subroutine and was edited to account for asymmetric tension/compression energy degradation. The resulting strength of the produced models have been presented as non-dimensional values. The finite element analysis (FEA) of the Matlab produced scaffolds showed that the fiber shifting between two adjacent layers, porosity, and voids of ellipsoidal shape that were perpendicular to the loading direction had the highest effect on the strength of the scaffold. The resulting normalized strength values obtained from the μ-CT models was partially validated through a comparison with the literature available.The different failure modes and overall architectural arrangement of cracks also showed promising results. / I den här uppsatsen så användes mikrotomografi (μ-CT) skanning av en bio-glas stödstruktur tillverkad av robocasting tekniken för att skapa finita element modeller med syftet att bestämma dess mekaniska styrka. Innan detta så användes ett Matlab-skript för att skapa flera förenklade geometrier av stödstrukturen i ett försök att fastställa betydelsen av viktiga designparametrar (som t.ex fiberpenetrering mellan två intilliggande plan) på stödstrukturens styrka. Vidare, för att bedöma påverkan av mikrostrukturella defekter som tomrum och mikrosprickor som är naturligt förekommande i robocasting-tillverkningsprocessen så uppmättes det totala antal hålrum och deras respektive volym med hjälp av μ-CT-skannade bilder. Denna data blev anpassad till en statistisk fördelning. Fördelningen av tomrum och mikcrosprickor användes sedan för att skapa flera modeller av stödstrukturerna i Matlab med antingen sfäriska eller ellipsoida hålrum närvarande. I de sista två modellerna undersöktes en en nedskalad och en uppskalad finita elementmodell baserad på μ-CT-skanning.För att modellera sprickinitiering, sprickpropagering och slutligen brott användes fasfältsmetoden. Fasfältsmetoden implementerades med hjälp av en för allmänheten tillgänglig Fortran användarrutin som redigerades för att ta hänsyn till en asymmetrisk energidegradering i drag-och tryck. Den resulterande styrkan hos alla modeller har presenterats som icke-dimensionella värden. Finita elementanalysen av Matlab modellerna visade att fiberskiftningen mellan två intilliggande plan, porositet och hålrum med ellipsoid form som var vinkelräta mot belastningsriktningen hade störst effekt på stödstrukturens styrka. De resulterande normaliserade styrkevärdena erhållna från μ-CTmodeller validerades delvis genom en jämförelse med tillgänglig litteratur. Dom olika felmoderna och övergripande strukturella fördelningen av sprickor visade också lovande resultat.