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

Are presented as mean sd n 3 denotes statistically

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Unformatted text preview: (A, C, and E) hydrogel matrices: (A and B) optical microscopy and (C and D) CLSM (F-actin in green, nuclei in blue) analysis of cell morphology. (E and F) cell viability (Live/ Dead assay: live cells in green, dead cells in red). Scale bars: A and B: 200 mM; C–F: 100 mM. (G) Cell metabolic activity (resazurin assay). (H) FRET-PVGLIG cleavage by MSC-secreted proteases present in CM (increase in fluorescence after 48 h). The Mann–Whitney test was used to compare two groups, ** p < 0.01, n ¼ 5. 4 Discussion The detailed analysis of the enzyme specicity of proteasecleavable motifs is essential to guide the design of proteolytically degradable hydrogel matrices. Here, the synthesis of a FRET sensor was vital to rapidly assess PVGLIG-cleavability in the presence of different MMPs and in heterogeneous solutions such as CM. PVGLIG was shown to be a good substrate not only for MMP-2 and MMP-9, as previously reported,19 but also for MMP-13 and MMP-14, with kcat/Km in the range of 105 MÀ1 sÀ1, characteristic of fast-degrading peptides.21 The comparison of This journal is ª The Royal Society of Chemistry 2013 the kcat/Km values calculated here with the ones reported by Chau et al.,19 suggests that our sequence has similar sensitivity to MMP-2 but higher sensitivity to MMP-9. In their work, the kinetic constants referred to dextran–PVGLIG–methotrexate conjugates and not to isolated PVGLIG, which may explain the observed differences.19 Moreover, the sequence tested here includes additional amino acids that may interfere with the substrate-binding specicity of active sites of the MMPs. We further demonstrated that the peptide could be cleaved by MMPs in soluble or coupled-to-alginate forms. It was interesting to uncover the MMP-14 (MT1-MMP)-sensitivity of the designed sequence, as this membrane-type protease plays a key role in pericellular proteolysis, either directly or indirectly through activation of other MMPs (like MMP-2).30 This tightly regulated and conned mechanism allows cells to accurately coordinate the dynamics of matrix degradation, not only in the native ECM30 but also in ECM-like articial matrices.17,31 The functional involvement of MMPs in a wide variety of hMSC activities, namely in invasion and differentiation, has Soft Matter, 2013, 9, 3283–3292 | 3289 View Article Online Downloaded by State University of New York at Buffalo on 16/04/2013 03:56:51. Published on 08 February 2013 on | doi:10.1039/C3SM27560D Soft Matter been well-documented.28,29,32 MMP-2, MMP-9, MMP-13 and MMP-14 have all been associated with bone remodelling, and several in vitro studies suggested the active participation of some of them in osteogenesis.29,32 Therefore, the prole of endogenous MMP expression in hMSC cultures under basal and osteogenic conditions was investigated in this study to elucidate their potential role in the degradation of PVGLIG, and justify further examination in 3D hydrogels. We have not detected the presence of MM...
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