Characterization of Bioactive Components in Decellularized Adipose Tissue Scaffolds for Tissue Engineering
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In previous in vitro and in vivo studies, decellularized adipose tissue (DAT) has demonstrated unique bioactivity, but little is known about the bioactive components preserved in the decellularized scaffold. With the goal of characterizing the bioactive components in the DAT, protein was extracted from DAT samples from 3 donors using 5 different buffers. The resulting DAT extracts were found to have very low protein content so molecular weight fractioning centrifugation was used to concentrate the samples. Concentrated extracts were screened for the presence of the bioactive components adiponectin, vascular endothelial growth factor A (VEGF-A), bone morphogenetic protein 2 (BMP-2) and Dickkopf related protein 1 (DKK-1) using Western blotting. Positive signal for BMP-2 was found for one donor in Roger’s Sample Buffer and Urea Buffer, but all other proteins investigated with Western blotting went undetected for all extraction buffers. Immunohistochemistry (IHC) was also used to determine the presence and distribution of bioactive components in the DAT samples. Sections of DAT were probed for the same bioactive components; adiponectin, VEGF-A, BMP-2 and DKK-1, with positive signals detected for adiponectin and VEGF-A. In order to develop an injectable, DAT-derived hydrogel for soft tissue regeneration, DATgels were fabricated from an enzymatically treated and homogenized DAT pre-gel suspension, which was neutralized to physiological pH and salt concentration. The highly-hydrated DATgels, containing up to 97% water, were found to degrade substantially over 14 days in simulated physiological fluid, with only a slight reduction in the overall scaffold size. Histology and SEM showed no major structural differences between the two formulations evaluated (40 mg/mL and 50 mg/mL), with both containing intact DAT features and small void spaces scattered heterogeneously throughout the scaffold. Preliminary in vitro cell work with adipose-derived stem cells (ASCs) showed that the 40 mg/mL formulation DATgel supported cell attachment and viability greater than 70% for all scaffolds up to 7 days after seeding. Further, migration into the scaffold was observed over time, indicating that the adhesive properties of the native ECM were retained through the processing steps required to fabricate the DATgels.