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

Abstract

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

Functionalising Collagen-Based Scaffolds With Platelet-Rich Plasma for Enhanced Skin Wound Healing Potential

“…As before, the composite scaffold was kept in medium without FBS or PRPr supplementation. As expected (Mcfadden et al, 2013;Do Amaral et al, 2019), collagen-GAG scaffolds were properly vascularised after 10 days of culture ( Figure 8A). By qualitatively analyzing the immunofluorescence images, the use of PRPr in collagen-GAG medium supplementation did not greatly impact scaffold vascularization compared with FBS supplementation (Figure 8B).…”

“…In order to fabricate collagen-GAG scaffolds, microfibrillar type I bovine tendon collagen (Integra Life Sciences, Plainsboro, NJ) was blended with chondroitin-6-sulfate, isolated from shark cartilage (Sigma-Aldrich, Germany) in 0.05 M acetic acid and freeze-dried as previously described (O'brien et al, 2005;Murphy et al, 2010;Do Amaral et al, 2019). Briefly, the suspension was frozen to a final temperature of −10 • C, which was maintained constant for 60 min, and then sublimated under vacuum (100 mTorr) at 0 • C for 17 h. The resulting porous scaffolds were cut into discs of 4 × 6 mm, physically crosslinked by a dehydrothermal treatment (DHT) using a vacuum oven (Vacucell, MMM Group, Munich, Germany) at 0.05 bar and 105 • C over 24 h, followed by a chemical crosslinking using 1-ethyl-2-(3-dimethylaminopropyl) carbodiimide (EDAC) in combination with N-hydroxysuccinimide (NHS) as previously described.…”

“…To evaluate the scaffolds capacity to be vascularised in vitro, a co-culture system of HUVEC and hMSC in a ratio of 4:1, respectively, with 5 × 10 5 cells in total was performed as previously described, with minor modifications (Duffy et al, 2011;Mcfadden et al, 2013;Lloyd-Griffith et al, 2015;Do Amaral et al, 2019). HUVEC were initially seeded with hMSC added to the culture 3 days after.…”

“…Three images were taken per sample, in which 1 was from the center and 2 from the periphery. Vessel-like structures were manually counted and automatically measured (area and ferret) using an in-house developed and publicly available ImageJ macro (Do Amaral et al, 2019). Statistical significance was determined by One-way Anova with Tukey's Multiple Comparison Test.…”

Blood and Brain Gene Expression Trajectories Underlying Neuropathology and Cognitive Impairment in Neurodegeneration

“…Common functional pathways CCKR (associated with appetite control and body weight regulation (Perry and Wang, 2012)), PDGF (with a significant role in blood vessel formation (Amaral et al, 2018), B cell activation (involved in immune system response), and VEGF and angiogenesis (linked to the formation of new blood vessels), evidenced the direct relationship between the central nervous system and the body, both in health and in disease. Their unsupervised datadriven identification is, therefore, supporting the crucial importance of studying the periphery-brain axis (e.g.…”