Recombinant human mmp 2

Enzyme-responsive release of TA from TG-18 hydrogel was performed at pH 7.4 and 37 °C. TA-loaded hydrogels (50 μl, 20 mg TA/ml) were placed in dialysis tubing (8–10 kDa molecular weight cut-off; Spectrum Labs) and suspended in PBS (950 μl) without or with one of the following enzymes: esterase (T. lanuginosus lipase, 200, 400, or 800 U/ml) (Sigma Aldrich); recombinant human MMP-2 (1.5 μg/ml) (Sigma Aldrich); recombinant human MMP-3 (5 µg/ml) (Sigma Aldrich), and recombinant human MMP-9 (1 μg/ml) (Sigma Aldrich). In some experiments, MMP-2/9 Inhibitor II (Sigma Aldrich) or MMP-3 Inhibitor II (Sigma Aldrich) was added along with the MMPs. Fresh enzyme or enzyme + MMP inhibitor were added at multiple time points as indicated in the figure legends. The dialysis bags filled with hydrogel in release medium were placed in 45 ml sink medium (PBS), and incubated at 37 °C with a shaking speed of 150 rpm. At each time point, an aliquot (1 ml) of sink medium was removed and replenished with the same volume of fresh PBS to ensure constant sink conditions. Aliquots were lyophilized and dissolved in 250 μl methanol, followed by high-performance liquid chromatography (HPLC) (Agilent 1100 quaternary LC pump liquid chromatograph, Zorbax SB C-18 column, 250 × 4.6 mm, 5 µm).

Gelatin from bovine skin type B (gel strength 225 g Bloom), protease from Streptomyces griseus, NaOH, MMP-2 human recombinant, glyoxal solution 40% and glycine were purchased from Sigma-Aldrich (St. Louis, MO, USA). TM and HPMC were obtained from Fagron (Barcelona, Spain). Absolute ethanol, trifluoroacetic acid and acetonitrile were acquired from Panreac (Madrid, Spain). Dialysis membrane with a molecular weight cut-off 3500 g/mol was provided by Medicel Membranes (London, UK). Timabak 0.5% (Thea Laboratories; Madrid, Spain) was purchased in a local pharmacy store. Ultrapure milli-Q water was used in all the studies. All other chemicals were reagent grade and used as received.

Gelatin zymography was used to evaluate the MMP-2 activity in the cardiac tissue. The protein concentration was determined by the Bradford method, and then, 70 μg protein was loaded to the 8% sodium dodecyl sulfate- (SDS-) polyacrylamide gel copolymerized with gelatin (20 mg/mL; type A from porcine skin; Sigma). Following electrophoresis, the gels were washed with 2.5% Triton X-100 and incubated overnight at 37°C in an incubation buffer containing 50 mM Tris-HCl, 150 mM NaCl, and 5 mM CaCl₂. A staining method was performed by using 0.05% Coomassie Brilliant Blue, and then, a mixture of 4% methanol and 8% acetic acid was added to the gels. Following washing procedures, the gels were digitally scanned and analyzed by using Quantity One software. MMP-2 human recombinant (Sigma-Aldrich, Hungary) was used as a positive control to identify MMP-2 activities, which were expressed as intensity × mm² [16 (link)].