Platelet-derived growth factor stabilises vascularisation in collagen-glycosaminoglycan scaffolds in vitro

Collagen-glycosaminoglycan (CG) scaffolds have been widely developed for a range of regenerative medicine applications. To enhance their efficacy, CG scaffolds have previously been prevascularised in vitro using human umbilical vein endothelial cells and human mesenchymal stromal cells (hMSCs); however, at later timepoints, a regression of vascularisation is observed. This is undesirable for longer preculture periods (e.g., for partial/full organ regeneration) and for in vitro vascularised tissue model systems (e.g., for drug testing/modelling). We hypothesised that delayed platelet-derived growth factor (PDGF)-BB addition could stabilise vessels, preventing their regression. In 2D, we identified 25 ng/ml as a suitable dose that enhanced hMSC metabolic activity and proliferation, without affecting endothelial cells, or migration in either cell type. In our 3D model of CG scaffold vascularisation, early addition of PDGF (Day 3) behaved similarly to no PDGF controls. However, PDGF addition at later timepoints (i.e., Days 4 and 5), with a second addition on Day 10, prevented vascular regression. In quantifying our observations, we identified a need for a tool to measure in vitro vascularisation in porous scaffolds. This was a second key objective of this work. A novel ImageJ macro was developed, which allowed us to analyse vessel-like structures, evaluating their number and morphology, and confirmed our qualitative observations. Finally, upregulation of angiogenic genes (ANG1, KDR, and TEK2) involved in vessel maturation illustrated how PDGF addition contributed to vascular stability. Taken together, the results suggest that addition of PDGF at specific timepoints can be used to stabilise vasculature in CG scaffolds.