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

In vitro studies suggested the active participation

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Unformatted text preview: P-9, which is consistent with other studies, namely by of Ries et al. who showed that MSC constitutively express MMP-2 (and MMP-14) activity, but only express MMP-9 when stimulated with specic cytokines.28 MMP-13 was also not detected. This may be related to the duration of the culture (3 weeks), which might have been too short given that MMP-13 is maximally expressed at later stages of osteoblast differentiation.33 It is also possible that MMP-13 could eventually be present at undetectable amounts, since Barthelemi et al. for example showed that levels of secreted MMP-13 in human osteoblasts CM were on average 50-fold lower than those of MMP-2.29 Nevertheless, it is important to highlight that, although not detected under the present in vitro conditions, MMP-9 and MMP-13 are likely to play a relevant role in the degradability of MMP-sensitive hydrogels, namely in vivo. For example, in an implantation scenario, the hydrogel will interact with different inammatory cells that not only produce MMP-9, but also secrete cytokines and growth factors known to stimulate MMP-9 secretion by MSC.28 In contrast, signicant levels of MMP-2 activity were detected throughout the culture, in both OM and BM. MMP-14 was not detected by zymography, as it would be expected for a membrane-anchored MMP, but in accordance with the current understanding of MMP-2 proenzyme activation, the presence of active MMP-2 in CM indirectly suggests that hMSC also expressed MMP-14 activity.17 Given the central role that MMP-2 and MMP-14 are expected to have in our system, particularly under in vitro conditions, we additionally characterized their expression at the mRNA levels. Although post-transcriptional regulatory processes could further alter MMP protein amounts, the mRNA expression of MMP-2 followed a pattern similar to its secretion prole, being lower in OM in agreement with other reports.34,35 In contrast, MMP-14 mRNA levels were increased in osteogenic cultures. Recently, the involvement of several MMPs in the differentiation of human osteoblasts in 3D collagen matrices was investigated,29 and a clear relationship between MMP-14 expression and osteogenesis was demonstrated. In fact, MMP-14 was crucial for ALP activity regulation, and silencing of this protease almost abolished osteocalcin expression.29 Here, we also observed that under osteogenic conditions MMP-14 expression and ALP activity had a synchronized peak around day 7. In addition to MMP proling, we also evaluated the global activity of the MSC secretome in its ability to cleave the target PVGLIG peptide. The cocktail of proteases (likely rich in MMP-2) present in CM from both undifferentiated and osteogenic MSC cultures was in fact able to degrade the soluble FRET-PVGLIG, but not the scrambled peptide. Aer the synthesis of water-soluble PVGLIG–alginate conjugates, these were used in the preparation of MMP-sensitive 3290 | Soft Matter, 2013, 9, 3283–3292 Paper hydrogel matrices for MSC entrapment. Cell behaviour in...
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