Characterization and Cell-Seeding of Decellularized Adipose Tissue Foams for Wound Healing

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Date
2014-09-17
Authors
Turco, Bryen
Keyword
decellularized adipose tissue , adipose-derived stem cell , wound
Abstract
Chronic wounds are common among diabetic patients and they are a leading cause of foot amputations. The standard of care has not been successful in healing these wounds so alternative cell-based approaches are being investigated. Each cell-based approach necessitates a delivery strategy that minimizes cell death and maximizes cell retention. To this end, an adipose-derived stem cell (ASC) delivery system was developed, using an extracellular matrix (ECM)-derived bioscaffold as the platform. Porous decellularized adipose tissue (DAT) foams were fabricated over a range of concentrations, and with different tissue processing methods (mincing vs. milling). These foams were characterized by quantitative assessment of equilibrium water content, porosity, protein loss, and swelling ratio (n = 3), with the aim of elucidating how protein concentration and tissue processing affect foam properties. DAT foams were hydrophilic, porous, and maintained their form in culture despite the gradual loss of protein. Also, increasing DAT foam concentrations resulted in reduced porosity and equilibrium water content. Immunofluorescence was then used to detect extracellular matrix (ECM) constituents including collagen IV, laminin and fibronectin in DAT foam cross-sections. DAT foams were positive for all three ECM proteins, suggesting that they could allow cell attachment, migration, and proliferation. In a previous study, it was identified that ASC do not infiltrate DAT foams using traditional static cell-seeding techniques. Therefore, cell-seeding studies were performed in an attempt to enhance cell infiltration into the central regions of the foams. dsDNA content and cell density were quantified in response to various seeding methods, scaffold concentrations, and orbital shaker speeds until an improved set of seeding conditions was identified. Proliferation of ASCs in foams was then determined by semi-quantitatively evaluating Ki67 expression and dsDNA content over 14 days. Finally, to determine the effects of cell-scaffold interactions on ASC gene expression, cells were seeded onto various substrates and cultured in normoxic (21%) or hypoxic (1%) conditions. mRNA levels of VEGF-A and FGF-2 were quantified using qRT- PCR, and it was found that there were no noticeable trends in terms of the effects of oxygen tension or cell-scaffold interactions. However, protein analysis should be performed to confirm these trends.
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