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

Direct contacts with neighbouring cells fig 6b and d

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Unformatted text preview: nce of CM from hMSC cultures within PVGLIG/RGD–alginate, as compared to RGD–alginate. Upon induction, hMSC were able to differentiate along the osteoblastic lineage in both types of matrices. As depicted in Fig. 7A and B (cytostaining and colorimetric assays, 3288 | Soft Matter, 2013, 9, 3283–3292 Fig. 5 Physico-chemical properties of binary alginate hydrogels composed of 50% LMW alginate (grafted or not with PVGLIG) and 50% HMW alginate (grafted or not with RGD) analyzed by DMA (viscoelastic properties) and cryoSEM (microstructure): (A) E0 and tan d as a function of the amount of PVGLIG grafted to the LMW fraction (L0 to L200). (B) E0 and tan d values of the hydrogel formulations used in the cell culture studies, loaded or not with MSC, 24 h after crosslinking. Results are presented as mean Æ SD (n ¼ 5) and are representative of at least 3 independent experiments. No statistical differences were found between the tested groups. (C) Microstructure of MSC-laden hydrogels at 24 h post-entrapment illustrates the similarities between the samples. respectively), ALP activity was greatly increased aer 21 days in OM vs. BM, and was slightly higher in MMP-sensitive matrices. As expected, hMSC remained metabolically active aer 3 weeks of culture (Fig. 7C) under all the tested conditions. Zymography analysis of CM showed that under all tested conditions the secretion of MMP-2 decreased from day 7 to day 21 (Fig. 7D). Only a slight decrease of MMP-2 activity in OM compared to BM was observed. However, and most importantly, the amounts of both latent and active MMP-2 forms were mainly up-regulated in PVGLIG/RGD–alginate matrices, compared to RGD-matrices at these time points. This journal is ª The Royal Society of Chemistry 2013 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 http://pubs.rsc.org | doi:10.1039/C3SM27560D Paper Soft Matter Fig. 7 3D-culture of MSC within MMP-sensitive (PVGLIG/RGD–alginate) and MMP-insensitive (RGD–alginate) hydrogels under osteogenic (OM) vs. basal (BM) conditions: (A) ALP activity, showing the positive staining for both hydrogels (representative picture of BM for both hydrogels is presented, original magnification 63Â) and (B) ALP activity quantification at day 21 is significantly higher for both samples in OM compared to BM; (C) cell metabolic activity at day 21 in relation to day 1 (resazurin assay). (D) MMP-2 activity in 3D-culture supernatants from days 7 and 21 (gelatine-zymography). Data are representative of at least 2 independent experiments. Quantitative results are presented as mean Æ SD (n ¼ 3). * denotes statistically significant differences (p < 0.05), as determined by the non-parametric Kruskal–Wallis test, followed by Dunn's post-test of the selected groups. Fig. 6 Behaviour of MSC after 1 week of 3D-culture within MMP-sensitive (PVGLIG/RGD–alginate) (B, D, and F) and MMP-insensitive (RGD–alginate)...
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