INTRODUCTION: Vascular endothelial growth factor (VEGF) is the principal regulator of angiogenesis. Tightly bound gradients to extracellular matrix determine the microvascular endothelial cells fate during organogenesis and tissue regeneration and uncontrolled expression can lead to abnormal vascular growth and vascular tumours 1. Despite advances in tissue engineering, tight spatio-temporal control of VEGF remains a challenge hindering its therapeutic application. Nanoclay-gels have established potential in tissue engineering due to their capacity to sequester proteins for sustained and localised bioactivity 2, 3. The current study reports a biomimetic method to applying self-assembling nanoclay-gels comprising 3D gradients of VEGF to support localised spatio-temporal formation of microvasculature.
METHODOLOGY: Hydrous suspension of Laponite®, a synthetic smectite clay, were added to a solution containing biomolecules to facilitate the structured gels assembly via reaction-diffusion. The assembled structures were loaded with punctured VEGF gradients. Its spatial distribution and concentration were confirmed with fluorescent imaging and ELISA, respectively. The biocompatibility and bioactivity were assessed with a human umbilical vein endothelial (HUVEC) tubule formation assay and a 28-day murine subcutaneous implantation. A contrast agent was injected to visualise the new blood vessel formation via µCT and corroborated with immuno-staining.
RESULTS: Structured gels were able to controllably pattern the distribution of VEGF in 3D with a resolution of 40 - 120µm depending on assembly conditions. Patterned gels supported tubule formation of HUVECS grown on gel surfaces and µCT analysis of the in vivo study indicated vascularization of gels within regions of VEGF patterning. This was confirmed by histological analysis showing progressive cell invasion and degradation of the gel, and microvessel formation within the projected area of the VEGF pattern.
CONCLUSIONS: Clay-based structured gels are a promising delivery system of growth factors for tissue engineering applications with clinical relevance. Here we demonstrated for the first time its capacity to hold VEGF gradients for localized bioactivity.
1. Gianni-Barrera, R. et al. Stem Cells Transl Med. 9, 433-444 (2020).
2. Dawson J, I. et al. Adv Mater. 23, 3304 - 3308 (2011).
3. Page D, J. et al. Acta biomater. 100, 378-387 (2019).