Joris van Dongen

287 Adipose ECM hydrogels to release paracrine factors thickness was calculated with the difference of the two positions. Subsequently, the hydrogels were deformed by 20% and the deformation was held constant for 100s while the force response was monitored over time 27,28 . Force was converted in stress by dividing with the area of cross-section of the plunger, whereas the deformation was converted in strain (deformation/100).The slope of the straight-line plot between stress and strain during the deformation was taken as stiffness ( E ). Then the strain was held constant at 0.2 for 100 s and the stress was monitored. Since hydrogels are viscoelastic in nature the stress did not remain constant but decreased (relaxed) with time. Total relaxation in 100 seconds was also recorded. Relaxing stiffness ( E(t) ) was calculated by dividing relaxing stress with constant strain. The relaxationwas understood in terms of a generalized Maxwell model by fitting the experimental data with the equation using the optimization routine in the solver Add-in of Microsoft Excel 2007. Fitting started with one Maxwell element and gradually more elements were added till the decrease in chi-squared (error function) value became insignificant. Each Maxwell element was characterized by the stiffness ( E i ) and relaxation time constant ( t i ) for which it remained active (Fig. 5 B, C, D). The stiffness values were converted into relative importance using the equation RI 1 =100*E 1 /(E 1 +E 2 ….. E n ) . Table 1. Nomenclature of NAM and DAM hydrogels derived from different pre-gel solutions. Donor 1 Donor 2 Donor 3 Pre-gel solution A NAM1A/DAM1A NAM2A/DAM2A NAM3A/DAM3A Pre-gel solution B NAM1B/DAM1B NAM2B/DAM2B NAM3B/DAM3B Pre-gel solution C NAM1C/DAM1C NAM2C/DAM2C NAM3C/DAM3C NAM = non-diabetic acellular matrix; DAM = diabetic acellular matrix Immunoassay of ECM hydrogels NAM and DAM hydrogels (n=3) were produced as previously described for the immunoassay (two technical replicates) (2.6). Multiplex immunoassaying (Luminex, R&D systems) was used to measure release of eleven representative ASC-released proteinaceous factors according to the manufacturer’s protocol: vascular endothelial growth factor A (VEGF-A), angiopoetin-1 (Ang-1) and angiopoetin-2 (Ang-2), matrix metalloproteinase 1 (MMP-1), tissue inhibitors of metalloproteinase 1 (TIMP- 1), interleukin-1 β (IL-1 β ), IL-6, IL-8/CXCL8, fibroblast growth factor 1 (FGF-1), hepatocyte growth factor (HGF) and monocyte chemotactic protein 1 (MCP-1/CCL2)).