Behaviour of endothelial cells in a tridimensional in vitro environment

Abstract

Angiogenesis is a fundamental process in healing, tumor growth, and a variety of medical conditions. For this reason, in vitro angiogenesis is an area of interest for researchers. Additionally, in vitro angiogenesis is important for the survival of prevascularized tissue-engineering models. The aim of this study was to observe the self-tubular organization behaviour of endothelial cells in the self-assembly method. In this study, bilayered and dermal substitutes were prepared using the self-assembly method. Histological, immunostaining, and biochemical tests were performed. The behavioural dynamics of endothelial cells in this biological environment of supportive cells were observed, as were the steps of the in vitro angiogenic cascade with self-organizing capillary-like structures formation. The epidermal component of the substitutes was seen to promote network expansion and density. It also increased the quantity of angiogenic factors (VEGF and Ang-1) without increasing the proinflammatory factor (IL-8). In addition, the increased MMP activity contributed to matrix degradation, which facilitated capillary formation.

Figure 1

Self-assembly method and experimental timelines. The skin substitutes were produced using the self-assembly method. The fibroblasts were cultured in the presence of ascorbic acid, and then ECs were seeded onto the fibroblast sheets. The manipulable sheets were transposed and incubated for 7 days to form the dermal component. Keratinocytes were seeded upon endothelialized fibroblast sheets to prepare bilayered skin substitutes. After one week of culture, the dermal and bilayered substitutes were raised to the air-liquid interface.

Figure 2

Endothelial cell purity and effect of conditioned medium from monolayer cultures. (a) Flow cytometry results of HDMECs. (b) CD31-Alexa 488 (green) IF staining of monolayer HDMECs (20x magnification, scale bar = 100 μm). Effect of conditioned media (CM) on endothelial cell monolayer culture organization and physiology on four-day-old culture. (c) Monolayer keratinocyte conditioned medium (KCM); circular branch-like structures were indicated with blue arrows. (d) Monolayer fibroblast conditioned medium (FCM). (e) Mixture of fibroblast and keratinocyte conditioned medium (MFKCM, 1 : 1). (f) EGM-2 MV SingleQuots Kit media with supplements and growth factors (EBM-2). (g) HDMECs proliferation assay with CM.

Figure 3

Histological analysis. Macroscopic appearance. (a) Bilayered skin model (epidermis + dermis). (b) Dermal model (without epidermis), histological analysis, and Masson's trichrome staining. (c) Bilayered skin model (10x magnification, scale bar = 100 μm). (d) Endothelialized bilayered skin model (20x magnification). (e) Ex vivo skin capillary (40x magnification, scale bar = 20 μm). (f) Dermal model (20x magnification, scale bar = 50 μm). Capillary-like tubes (CLTs) are indicated with arrows.

Figure 4

Observation of endothelial cell behaviour with whole-mount immunostaining. The steps of capillary formation in the in vitro skin model on a weekly basis after EC seeding: (a, b) fifth week, (c, d) sixth week, (e) seventh week, (f) eighth week, (g) lumen formation (40x magnification), and (h, i) ninth week, the end of the procedure (all figures (except (g)) were captured at 20x magnification with confocal microscopy) ((scale bar = 100 μm for (a), (b), (c), (e), (h), and (i)) – (scale bar = 50 μm for (f) and (g)) and scale bar = 200 μm for (d)).

Figure 5

Biochemical analysis with the conditioned media of substitutes. ELISA (n = 5 or 6), MMP activity (n = 6), and western blot results. ELISA results of (a) VEGF and (b) Ang-1. VEGF staining with IHC of (c) bilayered skin model and (d) ex vivo human skin (10x magnification, scale bar = 100 μm). (e) ELISA results of IL-8. (f) MMP activity analysis of bilayered and dermal substitutes and (g) collagen-I deposition of substitutes.