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

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Unformatted text preview: es: mRNA levels of MMP-2 and MMP-14, normalized to GAPDH. Results are presented as mean Æ standard error of the mean from 3 independent experiments (each using n ¼ 3 replicates). Although differences were not statistically significant, the same trend was observed in the 3 experiments. Table 2 samples Name L0 L10 L25 L50 L100 L200 Fig. 2 Activity of hMSC-secreted MMPs in conditioned media from basal and osteogenic conditions in 2D cultures: (A) gelatine-zymography: only MMP-2 was detected, and at higher amounts in BM compared to OM. Locations of the molecular weight (protein standards) on the zymogram are represented on the left. (B) FRET-measurements: MSC-secreted proteases were able to cleave the FRET-PVGLIG but showed little activity towards the scrambled peptide (control). MSC osteogenic differentiation was confirmed by (C) ALP and vK stainings at day 21 (scale bar ¼ 200 mm), and by (D) biochemical assay for ALP quantification. Results are representative of 3 independent experiments. Graphical results are presented as mean Æ SD (n ¼ 3). * denotes statistical significant difference from other groups, p < 0.05, as determined by the non-parametric Kruskal–Wallis test, followed by Dunn's post-test. conditions, but the opposite trend was found for MMP-14 expression (Fig. 3B), which seemed to be up-regulated. 3.3 Synthesis of soluble PVGLIG–alginate conjugates by carbodiimide chemistry LMW alginate was modied with different amounts of PVGLIG per gram of polymer. As summarized in Table 2, the amount of This journal is ª The Royal Society of Chemistry 2013 Summary of the compositions of PVGLIG-grafted LMW-alginate Coupling efficiency (%) Amount double-end graed (crosslinker, mg gÀ1) Amount single-end graed (tethered, mg gÀ1) 0 87.6 Æ 3.9 59.8 Æ 6.5 48.0 Æ 6.6 34.7 Æ 3.2 33.8 Æ 1.2 0 7.6 Æ 0.6 8.8 Æ 2.0 12.0 Æ 0.7 17.1 Æ 2.4 21.4 Æ 2.5 0 1.2 Æ 0.6 6.2 Æ 2.6 12.1 Æ 2.8 17.6 Æ 2.7 46.3 Æ 3.3 graed PVGLIG increased with the amount of peptide available for reaction but the graing efficiency decreased concomitantly. Additionally, the percentage of peptide that actually established links between alginate chains (double-end graed) also decreased when higher amounts of PVGLIG were used. In fact, ca. 87% of the total amount of coupled peptide was double-end graed in L10 (7.6 mg double-end graed PVGLIG per gram of alginate) while this only reached 32% in L200 (21.4 mg doubleend graed PVGLIG per g of alginate). A 20-fold increase of the amount of peptide available for reaction resulted in a less than 3-fold increase of the amount of “effective” double-end graed peptide. Overall, the best results (higher efficiency of coupling and double-end graing) were obtained with the L10 formulation, which was therefore selected for the cell culture studies. Incubation of PVGLIG–alginate conjugates with MMP-2 and MMP14 revealed that, upon graing to the alginate backbone, the PVGLIG sequence remained cleavable by these two MMPs, as...
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