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

Component the microstructure of msc laden matrices

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Unformatted text preview: rved using a JEOL JSM 6301F/Oxford INCA Energy 350/ Gatan Alto 2500 microscope. 2.3.4 Characterization of 3D hMSC cultures within peptide–alginate hydrogels. For these studies, MMP-insensitive RGD–alginate and MMP-sensitive PVGLIG/RGD–alginate Soft Matter, 2013, 9, 3283–3292 | 3285 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 hydrogels were used (n ¼ 3 per condition). Aer 1 week of culture under BM, hMSC morphology was imaged by brighteld optical microscopy and confocal laser-scanning microscopy (CLSM). For CLSM, F-actin was stained with Alexa Fluor 488 phalloidin (Molecular Probes, Eugene, OR USA), nuclei were co-stained with 1 mg mLÀ1 DAPI (40 ,6-diamidino-2-phenylindole) and samples were imaged in a Leica SP2AOBS microscope. Cell metabolic activity was monitored along the week using resazurin assays. For this, matrices were incubated in medium with 20 mg mLÀ1 resazurin for 2 h at 37  C and later the supernatants were transferred to a 96-well black plate with a clear bottom (Greiner) for uorescence quantication at lEx ¼ 570 nm and lEm ¼ 590 nm. Cell viability at day 7 was analyzed using the Live/Dead (Molecular Probes, Eugene, OR USA), according to the manufacturer's instructions. For MMP activity evaluation, CM was collected at day 7 aer 24 h of serum-starvation, centrifuged to remove debris and incubated with 5 mM of FRET-PVGLIG for 2 h at 37  C. The increase in uorescence with respect to the blank (CM without peptide) resulting from the FRET-PVGLIG cleavage was measured in a microplate reader at lEx ¼ 320 nm and lEm ¼ 420 nm. hMSC-laden matrices (n ¼ 3 per condition) were also cultured in OM for 3 weeks. The cell metabolic activity was checked at days 1 and 21, using the resazurin assay. To assess osteogenic differentiation, ALP activity was monitored at day 21 using a biochemical colorimetric assay and a whole-mount staining, both described above. For the colorimetric assay, cells were rst released from the matrix using 50 mM EDTA. ALPstained samples were imaged using a stereoscope with a digital camera (SZX10 and DP21, Olympus). CM from 3D-cultures maintained in BM or OM was collected at days 7 and 21, aer serum-starvation for 24 h, and MMP activity was analyzed by gelatine-zymography as already described. 2.4 Statistical analysis Statistical analyses were performed using GraphPad Prism 5.0 soware. Statistical signicance was determined using the nonparametric Kruskal–Wallis test, followed by Dunn's post-test. The non-parametric Mann–Whitney test was used to compare two groups. Differences were considered statistically signicant when p values were lower than 0.05. 3 Results 3.1 PVGLIG cleavage by different MMPs All the tested MMPs were able to efficiently cleave the nonspecic FRET-PLGL peptide (Fig. 1, inset) certifying the active state of the enzymes. When the same MMPs were in...
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This document was uploaded on 09/21/2013.

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