Un patient peut souffrir d’une perte de substance osseuse de taille critique suite à des accidents ou des pathologies. Aujourd’hui, le traitement le plus fréquent consiste en la greffe du tissu osseux (autogreffe ou allogreffe). Compte tenu des complication rencontrées (réponse immunologique, morbidité du site donneur), la recherche actuelle s’inscrit dans la recherche de synthèse d’un biomatériau bioactif favorisant la régénération osseuse. Ces matériaux devraient imiter les qualités de la matrice extracellulaire osseuse pour stimuler la formation osseuse.Les cellules souches mésenchymateuses jouent un rôle important du fait de leur capacité de prolifération et différentiation en ostéoblastes. Pour profiter de ce potentiel des cellules souches, il est nécessaire de comprendre comment contrôler leur comportement et mesurer l’impact du microenvironnement cellulaire sur la différenciation ostéogénique de ces cellules. Comme les cellules souches mésenchymateuses sont capables de différencier en plusieurs phénotypes différents, il est indispensable de les diriger dans la direction désirée. Plusieurs facteurs qui influencent le devenir cellulaire ont été identifiés, comme certains peptides bioactifs, des facteurs mécaniques comme la rigidité, ou la topographie de surface de matériaux.Dans la matrice extracellulaire naturelle du tissu osseux, les cellules souches mésenchymateuses sont entourées d’une variété de principes actifs, dont le plus abondant est le collagène I. Cette protéine s’assemble pour former des nanofibres qui présentent une nanomorphologie périodique avec une périodicité bien définie. La question posée au début de ce travail était: Cette structure a-t-elle un impact sur la différenciation des cellules souches?Pour étudier l’impact de la périodicité nanofibrillaire, nous proposons dans ce travail de recherche l’utilisation d’hélices modèles qui miment en partie la morphologie du collagène. Les hélices nanométriques auto-assemblées des surfactants gemini peuvent avoir un pas d’hélice et un diamètre similaires à ceux du collagène. La modulabilité de ces paramètres et la possibilité de modifier ces structures par des molécules bioactives permettent de moduler les caractéristiques des nanoobjets et d’étudier l’impact de ces nanomatériaux sur les cellules souches mésenchymateuses. / Tissue engineering is a field related to regenerative medicine which aims at replacing or regenerating a patient’s tissue, usually using a combination of cells and bioactive material designed to influence cell behaviour. In approaches for bone regeneration, human mesenchymal stem cells (hMSCs) are a common choice because of their ability to proliferate and differentiate into osteoblasts. Harnessing this potential requires biomaterials which promote osteoblastic differentiation, for example by mimicking the conditions in natural bone. Collagen I is a common protein in human bone; it forms fibrils with a characteristic periodic structure, which raises the question whether this morphology has in impact on stem cell fate. Collagen-mimicking nanomaterials can help investigate this question: Gemini surfactants with chiral counterions form twisted bilayers the morphology of which can be tuned by variation of enantiomeric excess, time and temperature. The self-assembled helical nanoribbons which are obtained by this process can be transformed by a sol-gel condensation to form silica nanohelices the size and twist pitch of which resembles that of collagen fibres. The objective of this study is to prepare 2D culture environments featuring these nanomaterals (with and without bioactive peptide functionalisation) to explore the effect of these materials on hMSC differentiation.Silica helices are fabricated by synthesis of surfactants with tartrate as counterion, and organic-inorganic transcription using a silica precursor compound. They can be modified by reaction with APTES and an N-hydroxysuccinimide ester and covalent immobilisation of a peptide. Two peptides were used in this study, one adhesion-promoting peptide and the active domain of the osteogenesis-inducing peptide BMP2. Helices with or without this bioactive functionalisation were covalently grafted to glass substrates using APTES and EDC/NHS-coupling. The presence of peptides on helices was shown by the absorption of helix-grafted peptides bearing the FITC-fluorophore. Successful peptide grafting onto glass surfaces was verified by XPS and fluorescence microscopy. The morphology of helices was monitored with TEM and SEM. SEM images were used to determine the amount of helices on surfaces. HMSCs were cultivated for four weeks on surfaces modified with APTES, peptide(s) or left- or righthanded nanohelices, functionalised or not with bioactive peptide(s). After fixation, the quantities of the osteogenic markers Runx2 and Osteocalcin (OCN) in the cells were evaluated. The results show that BMP2-functionalised surfaces exhibited an elevated level of Runx2 and OCN expression. Some helix-grafted materials exhibited a significantly higher Runx2 and/or OCN expression than the corresponding homogeneous materials, but these differences were not consistent across samples of the same chiral orientation or bioactive functionalisation. Therefore, conclusive general statements about differences in osteogenic effect between helix functionalisations and handednesses aredifficult to make. A potential reason for this is the variability of surface coverage of helix-grafted materials: As the quantity of helices that are immobilised onto the surfaces is lower than expected and varies greatly between the samples, the number of cells that are not in contact with the helices might change as well, which can lead to false negatives.The results of a proteomic experiment have shown which proteins are differentially expressed in cells cultured on helices with or without BMP-functionalisation, compared to bare glass. Comparison with other proteomic studies shows that proteins which are known to be upregulated during osteogenic differentiation are overexpressed most frequently in cells cultured on BMPmodified helices. The proteins that were identified with this method might serve as starting point for future investigations.
| Identifer | oai:union.ndltd.org:theses.fr/2017BORD0619 |
| Date | 16 June 2017 |
| Creators | Kemper, Gregor |
| Contributors | Bordeaux, University of Waterloo (Canada), Durrieu, Marie-Christine, Wettig, Shawn |
| Source Sets | Dépôt national des thèses électroniques françaises |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation, Text |
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