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

Peptide l0 was also prepared the graing eciency was

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Unformatted text preview: r assay and the percentage of double-end graed PVGLIG was quantied using uorescamine.17 The latter reacts with free amines generating a uorescent signal that can be measured at lEx ¼ 400 nm and lEm ¼ 460 nm. To probe the cleavability of PVGLIG–alginate conjugates by MMPs, these were incubated with 40 nM of activated MMP-2 and MMP-14 in TCNB buffer for 24 h at 37  C and cleavage was estimated using uorescamine. The cell-adhesion RGD peptide (GGGGRGDSP, GenScript) was graed to partially oxidized (1%) HMW alginate24 at a typical amount of 9 mg of graed RGD per gram of alginate (HMWRGD).17 2.3.2 Preparation of peptide–alginate hydrogel matrices. Lyophilized alginate powders were dissolved overnight in 0.9 wt % NaCl, sterile-ltered (0.2 mm), and then mixed with an aqueous suspension of CaCO3. Hydrogel formation was triggered by the addition of a fresh solution of d-gluconolactone (GDL). The Ca2+/COOÀ and Ca2+/GDL molar ratios were set as 0.36 and 0.50, respectively, as previously established.17,25 Hydrogel-precursor solutions were loaded in a QGelÔ Disc Caster and allowed to crosslink as small discs. Hydrogels were prepared at a nal alginate concentration of 2 wt%, with a previously optimized bimodal Mw composition of 50% LMW alginate and 50% HMW alginate, with or without graed peptides.14,17,26 For 3D culture, MSC were combined at 8 Â 106 cells per mL with the hydrogel-precursor solutions prior to crosslinking. Two hydrogel formulations were tested: 50% HMW-RGD plus 50% LMW-L0 (MMP-insensitive, hereaer designated RGD–alginate) and 50% HMW-RGD plus 50% LMW-PVGLIG L10 (MMP-sensitive, hereaer designated PVGLIG/RGD–alginate). Aer 30 min at RT, hMSC-laden hydrogels were transferred to 24-well suspension culture plates (Greiner), pre-treated with pHEMA (0.8 mg cmÀ2) to avoid cell adhesion to the bottom of the plates,27 and incubated in BM or OM under standard culture conditions at 37  C in a humidied atmosphere with 5% v/v CO2. 2.3.3 3D-matrix characterization: dynamic mechanical analysis (DMA) and cryoSEM. Swollen hydrogel samples (h ¼ 1.5 mm, B ¼ 6.8 Æ 0.3 mm), with or without cells, were tested under unconned compression using a Tritec 2000 DMA (Triton Technology, UK). Samples (n ¼ 5 per condition) were pre-equilibrated overnight at 37  C in DMEM supplemented with 25 mM HEPES and 0.01 wt% NaN3 at pH 7.5, and taken out of this solution only immediately before analysis. A small preload was used to promote an adequate contact between the sample and apparatus surfaces. A time-scan assay was performed at 1 Hz, 1% strain, for 5 min under controlled atmosphere (20  C and 44% humidity). DMA results are presented as storage modulus (E0 , elastic component) and tan d (corresponding to the ratio E00 /E0 , where E00 represents the loss modulus or viscous component). The microstructure of MSC-laden matrices was imaged 24 h post-entrapment by cryogenic scanning electron microscopy (cryo-SEM). Fractured surfaces of liquid N2-frozen samples were obse...
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This document was uploaded on 09/21/2013.

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