Synthesis and Characterisation of Chiral Nanomaterials and their Influence on Stem Cell Differentiation

Biomaterials;Mesenchymal stem cells;Silica nanostructures;Cell differentiation;Nanoperiodicity

Kemper, Gregor

University of Waterloo

关键词: Biomaterials;    Mesenchymal stem cells;    Silica nanostructures;    Cell differentiation;    Nanoperiodicity;   

Others  :  https://uwspace.uwaterloo.ca/bitstream/10012/12134/7/Kemper_Gregor.pdf

瑞士|英语

来源: UWSPACE Waterloo Institutional Repository

【 摘 要 】

Tissue engineering is a field related to regenerative medicine which aims at replacing orregenerating a patient’s tissue, usually using a combination of cells and a bioactive material whichis designed to influence cell behaviour in a desired way. In approaches for bone regeneration,human mesenchymal stem cells (hMSCs) are a common choice of cells because of their abilityto proliferate and differentiate into osteoblasts. Harnessing this potential requires biomaterialswhich promote osteoblastic differentiation, for example by mimicking the conditions in naturalbone. Collagen I is a common protein in human bone; it forms fibrils with a characteristic periodicstructure, which raises the question whether this particular morphology has in impact on stem cellfate. Artificial collagen-mimicking nanomaterials can help to investigate this question: Geminisurfactants with chiral counterions form twisted bilayers the morphology of which can be tunedby variation of experimental parameters like enantiomeric excess, time and temperature. The selfassembledhelical nanoribbons which are obtained by this process can be transformed by a solgelcondensation to form silica nanohelices the size and twist pitch of which resembles that ofcollagen fibres. The objective of this study is to prepare 2D culture environments featuring thesenanomaterals (with and without bioactive peptide functionalisation) in order to explore the effectof these materials on hMSC differentiation.Silica helices are fabricated by synthesis of surfactants with tartrate as counterion, and organicinorganictranscription using a silica precursor compound. They can be modified by reactionwith APTES and an N-hydroxysuccinimide ester and subsequent covalent immobilisation of apeptide. Two peptides were used in this study, one adhesion-promoting peptide featuring the RGD sequence and the active domain of the osteogenesis-inducing peptide BMP2. Helices with orwithout this bioactive functionalisation were covalently grafted to glass substrates using APTESand EDC/NHS-coupling. The presence of peptides on helices was shown by the absorption ofhelix-grafted peptides bearing the FITC-fluorophore. The successful peptide grafting onto glasssurfaces was verified by XPS and fluorescence microscopy. The morphology of helices wasmonitored with TEM before helix immmobilisation on surfaces, and with SEM afterwards. SEMimages were used to determine the amount of helices grafted to surfaces.HMSCs were cultivated for four weeks on surfaces modified with APTES, peptide(s) ornanohelices, the latter being either left- or righthanded and functionalised or not with bioactivepeptide(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 did indeedexhibit an elevated level of Runx2 and OCN expression. A cooperative osteogenic effect of RGDand BMP2 grafted together could be observed in terms of OCN, but not with Runx2. Somehelix-grafted materials exhibited a significantly higher Runx2 and/or OCN expression than thecorresponding homogeneous materials, but these differences were not consistent across samples ofthe same chiral orientation or bioactive functionalisation. Therefore, conclusive general statementsabout 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-graftedmaterials: As the quantity of helices that are immobilised onto the surfaces is lower than expectedand varies greatly between the samples, the number of cells that are not in contact with the helicesmight change as well, which can lead to false negatives.The results of a proteomic experiment have shown which proteins are differentially expressedin 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 upregulatedduring osteogenic differentiation are overexpressed most frequently in cells cultured on BMPmodifiedhelices. The proteins that were identified with this method might serve as starting pointfor future investigations.