| dc.description.abstract | 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 a bioactive material which
is 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 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 particular morphology has in impact on stem cell
fate. Artificial collagen-mimicking nanomaterials can help to investigate this question: Gemini
surfactants with chiral counterions form twisted bilayers the morphology of which can be tuned
by variation of experimental parameters like enantiomeric excess, time and temperature. The selfassembled
helical nanoribbons which are obtained by this process can be transformed by a solgel
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) in order to explore the effect
of these materials on hMSC differentiation.
Silica helices are fabricated by synthesis of surfactants with tartrate as counterion, and organicinorganic
transcription using a silica precursor compound. They can be modified by reaction
with APTES and an N-hydroxysuccinimide ester and subsequent covalent immobilisation of a
peptide. 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 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. The successful peptide grafting onto glass
surfaces was verified by XPS and fluorescence microscopy. The morphology of helices was
monitored with TEM before helix immmobilisation on surfaces, and with SEM afterwards. SEM
images were used to determine the amount of helices grafted to surfaces.
HMSCs were cultivated for four weeks on surfaces modified with APTES, peptide(s) or
nanohelices, the latter being either left- or righthanded and 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 did indeed
exhibit an elevated level of Runx2 and OCN expression. A cooperative osteogenic effect of RGD
and BMP2 grafted together could be observed in terms of OCN, but not with Runx2. 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 are
difficult 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. | en |
