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Unformatted text preview: 3D
microenvironments is largely inuenced by a number of matrixparameters, such as biochemical composition, mechanical
properties and porosity, which are oen interrelated and diﬃcult to tune in an independent manner. Hence, to ensure that
MSC behaviour in MMP-sensitive vs. MMP-insensitive hydrogels
would be mainly driven by diﬀerences in matrix susceptibility to
proteolytic degradation, it was essential to prepare hydrogels
with diﬀerent peptide-compositions but comparable viscoelastic and microstructural properties. In fact, since peptides
were graed to alginate carboxyl groups, which mediate the
ionic crosslinking of the hydrogels, their presence could interfere with the mechanism of hydrogel formation and alter the
nal physico-chemical properties of the matrices. Moreover, as
a crosslinking peptide, the presence of PVGLIG could further
contribute to the matrix mechanical properties. To overcome
this potential limitation, we devised a strategy that consisted in
preparing hydrogel matrices with a binary composition, where
the LMW fraction was modied (or not) with PVGLIG and the
HMW fraction was always modied with RGD (included at a
nal density of 130 mM to promote cell–matrix adhesion). The
rationale for this approach was based on prior knowledge that
the mechanical properties of binary alginate hydrogels are
mainly dependent on the HMW fraction.8 In fact, ionic crosslinking of 2 wt% alginate solutions with 100% LMW results is a
thick uid rather than a solid-like hydrogel. Therefore, small
changes in the composition of the LMW fraction (the maximum
amount of graed peptides represented less than 1.5 wt% of the
total polymer weight) do not result in signicant alterations of
the global properties of the hydrogels. This was eﬀectively
demonstrated here by DMA and cryoSEM analysis, which
conrmed that the peptide-composition of the 3D matrices
could be adjusted without concomitantly aﬀecting their stiﬀness and microstructure.
hMSC morphology in MMP-sensitive matrices was clearly
diﬀerent from that of cells in MMP-insensitive hydrogels.
Cleavage of PVGLIG-crosslinks in MMP-sensitive hydrogels by
MSC-derived proteases (most likely MMP-14 and MMP-2)
apparently created structural matrix-defects that conferred cells
some peripheral space to spread. As previously revealed by
zymography analysis, hMSC within PVGLIG/RGD–alginate
hydrogels secreted higher amounts of latent and active MMP-2
than cells in RGD–alginate matrices.17 Here, with the use of
FRET sensors, it was possible to verify that the secretome from
hMSC 3D cultures was eﬀectively active in cleaving PVGLIG, as
observed in 2D cultures. Moreover, this assay conrmed that
the presence of matrix-immobilized PVGLIG stimulated the
secretion of proteases (most likely MMPs) by entrapped hMSC
into the extracellular milieu.
In a pro-osteogenic environment, entrapped hMSC diﬀerentiated along the osteoblastic lineage in both types of hydrogels, and ALP activity slightly increased upon hMSC...
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This document was uploaded on 09/21/2013.
- Spring '13