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

Mmps recombinant human pro enzymes mmp 2 mmp 9 and

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Unformatted text preview: nneapolis, MN, USA) and active catalytic domains of MMP-1, MMP-8, and MMP-13 (Enzo Life Sciences, Exeter, UK) were reconstituted in TCNB (50 mM Tris, pH 7.5, 10 mM CaCl2, 150 mM NaCl and 0.05% v/v Brij 35). MMP-2 and MMP-9 were activated with p-aminophenylmercuric acetate (APMA) and MMP-14 was activated with Furin (R&D Systems, Minneapolis, MN, USA), as recommended by the manufacturers. Active MMPs were incubated with FRET-PVGLIG (5 mM) for 60 min at 37  C and the increase in uorescence resulting from peptide-cleavage was continuously monitored in a 3284 | Soft Matter, 2013, 9, 3283–3292 2.2 MMP proling in hMSC cultures 2.2.1 Culture and differentiation of hMSC. Human MSC were purchased from Lonza (PT-2501, Lot Nr 6F4392, Walkersville, MD, USA) and used between passages 5 and 7. hMSC seeded at 3 Â 103 cells per cm2 were cultured in a basal medium (BM): low-glucose Dulbecco's Modied Eagle Medium (DMEM with glutamax), 10% v/v MSC-qualied fetal bovine serum (FBS) and 1% v/v Penicillin/Streptomycin (P/S), all from Gibco (Paisley, UK); or an osteogenic medium (OM): low-glucose DMEM, 10% v/v FBS (pre-selected batch, PAA – Pasching, Austria), 1% v/v P/S, 100 nM dexamethasone, 10 mM b-glycerophosphate and 0.05 mM 2-phospho-L-ascorbic acid. Osteogenic differentiation was monitored through ALP activity and von Kossa (vK) stainings. Aer xation with 4% v/v paraformaldehyde (PFA) in PBS for 20 min, the cells were incubated for 30 min in Naphthol AS-MX phosphate/Fast Violet B salt at 37  C in the dark for ALP, or incubated in 2.5 wt% silver nitrate for 30 min under UV light, and then in 5 wt% sodium thiosulfate for 3 min for vK. Aer washing, the stained monolayers were air-dried and observed under an inverted microscope (Axiovert 200 M, Zeiss). Biochemical analysis of ALP activity was assayed in cell lysates. Samples were incubated with 2 mM pnitrophenol phosphate in 0.2 M bicarbonate buffer (pH 10), 0.05% v/v Triton X-100, and 4 mM MgCl2 for 1 h at 37  C. Absorbance was read at 405 nm aer quenching with 1 M NaOH. The amount of product was obtained from a standard curve prepared with serial dilutions of p-nitrophenol, normalized to the total cell protein content and converted into specic ALP activity (nmol per min per mg protein). 2.2.2 hMSC-secreted proteases: gelatine-zymography and cleavage of FRET-peptides. Conditioned media (CM) from hMSC cultures were collected at different time points (days 1, 7, 14 and 21) aer 24 h of serum-starvation and centrifuged to remove cell debris. Samples were loaded into 10 wt% 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 gelatine–SDS polyacrylamide gels and run in 1Â Tris–glycine SDS buffer at 60 V (Mini Protean Tetra Cell system, BioRad). Loading volumes were pre-adjusted according to the total protein content of cell l...
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