Joris van Dongen

292 Chapter 12 Hydrogels bind and release ASC-secreted factors Hydrogels derived from NAM and DAM samples mixed with concentrated CMe from cultured ASC bind released factors from ASC and had released part of the measured factors after 24h. The concentrations of factors were lower in the conditioned medium derived from NAM and DAM samples in comparison with the three baseline concentrations of ASC-CMe for MMP-1, CXCL8 and IL-6. The difference in concentration of factors indicates that the release of bound factors extends well beyond 24h. For Ang-1, Ang-2 and FGF-1 a higher concentration in conditioned medium derived from hydrogels as compared to the baseline concentrations was found. This implies that the hydrogels release more factors than initially were mixed in with ASC- CMe. This indicates that there were still factors present in ECM after decellularization that were detectable by the immunoassay, albeit that their bioactivity might not be retained after the harsh decellularization procedures. There was no significant difference in release pattern of any of the measured factors between non-diabetic and diabetic-derived hydrogels (p>0.05) (Fig. 3). The concentrations of IL-1 β and HGFwere below the detection limit. In contrast, TIMP-1 concentrations exceeded the maximal detection limit and were therefore not plotted. These high concentrations of TIMP- 1 indicate that most of the MMP-1 released by ASC and, therefore, released by the hydrogels is likely rendered inactive by binding of TIMP-1. Moreover, large interdonor variations in releasing patterns were observed, especially in diabetic derived hydrogels and mainly when two and four times concentrated ASC-CMe was mixed with the gels. This indicates a donor dependent binding capacity limitation of specific factors such as VEGF-A, Ang-1, Ang-2, MMP-1 and CCL2. Similar PK84 fibroblast migration when treated with released factors by CMe-loaded NAM and DAM hydrogels In comparison to serum free controls, conditioned medium from ASC (ASC-CMe1) promoted closure of damaged fibroblast monolayers (Fig. 4). Similarly, factors released from either NAM or DAM gels, promoted closure even after 96h of release (Fig. 4). A similar migration speed was detected between fibroblasts treated with factors from CMe-loaded NAM and DAM hydrogels released after 24h, 48h as well as 96h (p>0.05) (Fig. 4). However, in comparison to ASC-CMe1, factors from CMe-loaded DAM hydrogels released after 24h and 96h showed a smaller decrease in scratch surface area (p<0.05). For the NAM groups, all groups showed a larger decrease in scratch surface area in comparison to controls i.e. serum free culture medium (p<0.05). For the DAM group, released factors from CMe-loaded hydrogels after 48h and 96h in comparison with serum free controls showed a larger decrease in scratch surface area (p<0.05). This suggests that acellular adipose tissue matrix hydrogels released factors for at least 96h as observed by stimulated migration of fibroblasts.