Speaker
Description
"Introduction Tissue engineering (TE) is an interdisciplinary field that creates functional biologic substitutes for the repair of damaged tissues or organs. One major challenge when generating a functional TE model is its vascularization. Indeed, nutrient and oxygen supply, as well as metabolic waste products collection, are essential for the survival of the engineered tissue after transplantation. Hence, the development of a stable and functional vascular network within the constructs is essential. Using materials that include angiogenic cues as scaffolds for TE constructs may be a potential solution. Understanding this, the stromal vascular fraction (SVF) of adipose tissue has been proposed as a tool for in vitro pre-vascularization1. SVF is a heterogeneous cell population that has shown spontaneous vasculogenesis when cultured in vitro, in the absence of added growth factors. The extracellular matrix (ECM) produced by SVF cells is a key component to this capability. Many recent reports detail the use of ECM-derived hydrogels as TE scaffolds able to support cellular activities due to their similarity to native tissue’s ECM. We herein report the development of an angiogenic hydrogel derived from the ECM of SVF cell sheets.
Methodology SVF cell sheets were subjected to a decellularization protocol by a combination of freeze-thaw cycles and a nuclease treatment. Then, the samples were freeze-dried and digested with an acidic pepsin solution, and hydrogel polymerization occurred after pH neutralization with 0.5 M NaOH and temperature increase up to 37ºC for 1h. DNA quantification allowed to assess decellularization efficiency, while circular dichroism (CD) allowed to verify protein secondary structure maintenance. Regarding the ECM-related protein content, SDS-PAGE and Western blot were used. ECM-derived hydrogels were also stained for the presence of nuclei and collagen by using, respectively, Hematoxylin and Eosin (H&E) and Sirius Red/Fast Green staining.
Results DNA quantification and H&E staining confirmed decellularization efficiency. Through CD technique, it was possible to detect the triple helix conformational structure typical of collagen, confirming conservation of protein structure after the extraction protocol. Protein analysis with SDS-PAGE and Western blot revealed high protein variety within the ECM extract, with type I collagen being the predominant one. This was also verified by Sirius Red/Fast Green staining.
Conclusions Overall, these results show that we were able to isolate ECM proteins from SVF cell sheets and successfully create an ECM-like hydrogel with a Freytes solubilization protocol. Ongoing studies are focused on the proteomic characterization of the hydrogel as well as on in vitro cell culture studies to confirm the angiogenic potential compared with commercial collagen hydrogels. If effective, the use and development of regenerative strategies based on angiogenic ECM-like hydrogels can lead to promising advances in the TE and regenerative medicine fields.
Acknowledgements: EU Horizon 2020 research and innovation program under the ERC grant CapBed (805411); IF/00347/2015.
- Costa, M. et al., Acta Biomater. 55, 131–143 (2017)"
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