Fonseca et. al. Enzymatic, physicochemical and biological properties of MMP-sensitive alginate hydro

Likely rich in mmp 2 present in cm from both

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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.

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