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

Of coupling and double end graing were obtained with

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Unformatted text preview: probed by the uorescamine assay (Fig. 4). 3.4 Effect of PVGLIG incorporation on the viscoelastic and microstructural properties of bifunctional hydrogel matrices To assess the effect of graed PVGLIG on the viscoelastic properties of alginate matrices, the composition of the LMW fraction of binary hydrogels was systematically varied from unmodied alginate (LMW) to alginate modied with increasing amounts of PVGLIG (L0 to L200). As shown in Fig. 5A, the coupling procedure per se resulted in insignicant Soft Matter, 2013, 9, 3283–3292 | 3287 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 Paper Fig. 4 Enzymatic cleavage of PVGLIG–alginate conjugates in the presence of MMP-2 and MMP-14 (40 nM), after 0 and 24 h of incubation at 37  C. PVGLIG– alginate without enzymes was used as control. Results are presented as mean Æ SD (n ¼ 3). * denotes statistically significant difference (p < 0.05), as determined by the non-parametric Kruskal–Wallis test, followed by Dunn's post-test of the selected groups. changes on E0 and tan d, as can be concluded by comparing “LMW” with “L0”. Increasing the amount of PVGLIG in the LMW fraction resulted in an apparent slight decrease of E0 and increase in tan d, but differences were statistically negligible. The viscoelastic properties of the hydrogel formulations used in the cell culture studies were also characterized Fig. 5B. Again, the incorporation of PVGLIG did not signicantly alter the value of E0 or tan d, both in acellular or hMSC-laden hydrogels. The E0 of hMSC-laden hydrogels (z10 kPa) was slightly lower than that of acellular hydrogels (z15 kPa), but tan d values remained unchanged. All the tested hydrogels were shown to be predominantly elastic (E0 [ E00 ). From these results, it was possible to conclude that the MMP-insensitive (RGD–alginate) and MMP-sensitive (PVGLIG/RGD–alginate) used in the subsequent cell culture studies presented equivalent viscoelastic properties, as envisaged, and also similar microstructures, as revealed by cryoSEM analysis (Fig. 5C). 3.5 Spreading and protease-secretion of hMSC entrapped in MMP-sensitive alginate hydrogels Aer 1 week of culture within PVGLIG/RGD–alginate and RGD– alginate 3D matrices, differences in the hMSC morphology were clear. While cells within RGD–alginate remained mostly round and individually dispersed in the matrix (Fig. 6A and C), hMSC entrapped within PVGLIG/RGD–alginate hydrogels were able to spread, extend cytoplasm protrusions and establish direct contacts with neighbouring cells (Fig. 6B and D). In both types of matrices, hMSC were mainly viable (Fig. 6E and F) and metabolically active (Fig. 6G). To check PVGLIG cleavability by hMSC-secreted proteases, CM from both cultures were incubated with the FRET sensor. As shown in Fig. 6H, higher PVGLIG-cleavage was observed in the prese...
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