Development of a Composite Collagen-Agarose Hydrogel to Enhance Cellular Growth and Angiogenesis for Tissue Engineered Tracheal Grafts
Joanna Weber, Lauren Drake, Zaid Muslim, M. Jawad Latif, Kostantinos Poulikidis, Faiz Bhora.
Nuvance Health, Danbury, CT, USA.
OBJECTIVE: Composite trachea grafts made of collagen supported by 3D-printed plastic scaffolds contain void spaces between construct layers. To enhance cellular infiltration and support graft integrity, we developed a composite collagen-agarose hydrogel that permeates the entire graft providing a continuous cell substrate from the external side of the graft through to the lumen. In this study, we assessed the capacity of this hydrogel to support in vitro angiogenesis. METHODS: Human endothelial cells and fibroblasts were mixed in a 1:3 ratio and suspended at 1x106cells/mL in the composite hydrogels. Constructs were assessed after 7 days of culture by live light microscopy, immunohistochemistry, and qPCR to assess cell morphology, tube formation, and gene expression levels of VEGF and αSMA. RESULTS: Composite gels with agarose concentrations of 0.125% and collagen concentrations of 1 and 2mg/mL allowed cells quickly adopt a spread morphology and self-assembled into elongated structures with lumen formation, indicative of early vessel formation. Gene expression of VEGF and αSMA were increased at day 7 compared control constructs from day 0. In addition, immunostaining for αSMA, a protein highly associated with tissue vasculature, was positive. Overall these indicators demonstrated that the cells present in the hydrogels were adopting an angiogenic phenotype.
CONCLUSIONS: Rapid and sustainable angiogenesis is a key principle for the success of tissue engineered graft uptake. We have developed collagen-agarose hydrogel blends that support in vitro angiogenesis and will be used in future implantation studies of our tracheal graft to enhance cellular ingrowth and incorporation into the surrounding tissues.
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