The impact of intercellular communication for the generation of complex multicellular prevascularized tissue equivalents

Abstract

In reconstructive surgery the use of prevascularized soft tissue equivalents is a promising approach for wound coverage of defects after tumor resection or trauma. However, in previous studies to generate soft tissue equivalents on collagen membranes, microcapillaries were restricted to superficial areas. In this study, to understand which factors were involved in the formation of these microcapillaries, the levels of the angiogenic factors vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) in the supernatants of the tissue equivalents were examined at various time points and conditions. Additionally, the influence of these factors on viability, proliferation, migration, and tube formation in monocultures compared to cocultures of fibroblast and endothelial cells was examined. The results showed that VEGF production was decreased in cocultures compared to fibroblast monocultures and the lowest VEGF levels were observed in endothelial cell monocultures. Additionally, the highest levels of IL-8 were observed in cocultures compared to monocultures. Similar results were observed for bFGF with lowest levels seen within the first 24 hr and highest levels in cocultures. VEGF and IL-8 were shown to promote endothelial cell viability, proliferation and migration and angiogenic parameters such as tube density, total tube length, and number of tube branches. Addition of VEGF and IL-8 to cocultures resulted in accelerated and denser formation of capillarylike structures. The results indicate that VEGF, IL-8, and bFGF strongly influence cellular behavior of endothelial cells and this information should be useful in promoting the formation of microcapillary-like structures in complex tissue equivalents. K E Y W O R D S angiogenic growth factors, coculture, HDMEC, intercellular communication, microvascular endothelial cells

Vascularization strategies in tissue engineering approaches for soft tissue repair

“…Under normal conditions, blood vessels and ECs are in a quiescent state, but ECs have the dynamic capability to quickly respond to pro-angiogenic signals, for example, in case of injury (Unterleuthner et al, 2017). Several growth factors can activate angiogenesis, such as: vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), transforming growth factor β-1 (TGF-β1), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), Interleukin-8 (IL-8), tumor necrosis factor (TNF-α), angiopoietin, hypoxia-inducible factor 1(HIF), and epidermal growth factor (EGF) (Aghaloo & Hadaya, 2017;Heller et al, 2020;Karppinen et al, 2019;Unterleuthner et al, 2017;Wang & Xu, 2020). Angiogenesis is an extremely important event during repair, since the injured tissue and newly formed tissue are deprived of oxygen and nutrients.…”

“…Endothelial cells express specific markers such as platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31), von Willebrand factor (vWF), and vascular endothelial (VE)-cadherin (Cheung et al, 2015;Costa-Almeida et al, 2014;Heller et al, 2020;Nishiyama et al, 2019;Unterleuthner et al, 2017). Immunohistochemical or immunofluorescence methods can detect the presence of ECs by binding antibodies to the cells expressing one or more of the above markers, thus providing important information with regards to vascularization in tissue constructs.…”

“…Analysis of the microvascular structures marked by an endothelial marker such as CD-31 can be performed through fluorescence microscopy (Costa-Almeida et al, 2015), epifluorescence microscopy (Weinandy et al, 2014), confocal laser scanning microscopy (Costa-Almeida et al, 2015;Dos Santos et al, 2019;Heller et al, 2020;Heller et al, 2016;Ren et al, 2014;Unterleuthner et al, 2017;Xiao et al, 2015), two-photon laser scanning microscopy (TPLSM) (Kniebs et al, 2020;Kreimendahl et al, 2017;Samal et al, 2015;Weinandy et al, 2014), or scanning electron microscopy (SEM) (Lu et al, 2019;Samal et al, 2015;Shen et al, 2017).…”

“…Tube formation can be visualized by a phase contrast inverted microscope over 4-24 h with maximum tube formation at 4-6 h (DeCicco-Skinner et al, 2014). The captured images can be analyzed by commercially available software to quantify different parameters representing the degree of tube formation, such as the length and/or number of the formed tubes, MASSON-MEYERS AND TAYEBI number of branch points, and loops/meshes (Heller et al, 2020;Irvin et al, 2014;W. C. Liu, Chen, Zheng et al, 2017).…”

“…However, one of the crucial problems associated with tissueengineered equivalents, especially large-sized ones, is the possible lack or delayed blood vessel supply after their implantation in vivo (Heller et al, 2020). Small tissue equivalents will likely be easily vascularized from the ingrowth of blood vessels from the surrounding host tissue after implantation in vivo and it is enough to maintain cell viability.…”