In vivo organized neovascularization induced by 3D bioprinted endothelial-derived extracellular vesicles

Abstract

Extracellular vesicles (EVs) have become a key tool in the biotechnological landscape due to their well-documented ability to mediate intercellular communication. This feature has been explored and is under constant investigation by researchers, who have demonstrated the important role of EVs in several research fields ranging from oncology to immunology and diagnostics to regenerative medicine. Unfortunately, there are still some limitations to overcome before clinical application, including the inability to confine the EVs to strategically defined sites of interest to avoid side effects. In this study, for the first time, EV application is supported by 3D bioprinting technology to develop a new strategy for applying the angiogenic cargo of human umbilical vein endothelial cell-derived EVs in regenerative medicine. EVs, derived from human endothelial cells and grown under different stressed conditions, were collected and used as bioadditives for the formulation of advanced bioinks. After in vivo subcutaneous implantation, we demonstrated that the bioprinted 3D structures, loaded with EVs, supported the formation of a new functional vasculature in situ, consisting of blood-perfused microvessels recapitulating the printed pattern. The results obtained in this study favour the development of new therapeutic approaches for critical clinical conditions, such as the need for prompt revascularization of ischaemic tissues, which represent the fundamental substrate for advanced regenerative medicine applications.

Extracellular Vesicles as Drivers of Immunoinflammation in Atherothrombosis

“…To increase delivery efficiency to specific tissues, EVs have been combined with biomaterial scaffolds (tissue engineering). For instance, loading EC-derived EVs with bioprinted 3D structures [ 338 ] or a sustained delivery of EPC-derived EVs from injectable hydrogels [ 339 ] to support neovascularization and ameliorate cardiac dysfunction after MI. Recently, a synthetic nanotechnology leveraged to mimic the procoagulant function (adhesion and aggregation) of native platelets on nanoparticles has been reported.…”

Lymphatic and Blood Endothelial Extracellular Vesicles: A Story Yet to Be Written

“…BEC-EVs were found to express EC markers VE-cadherin, PECAM-1, vWF, and ENG [ 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 ]. In addition, angiogenesis-implicated growth factor receptors VEGFR-1 and VEGFR-2 and their ligand VEGF-A were detected, with the latter’s expression being increased under hypoxic conditions [ 52 , 62 , 63 ]. VE-cadherin expression on BEC-EVs is also primed under hypoxia [ 55 , 57 ].…”

“…Underlined bold molecules indicate parental cell markers. Common EV markers [ 5 , 42 , 46 , 77 ]; ECFC EV markers [ 29 , 48 , 49 , 50 , 51 ]; BEC EV markers [ 44 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 61 , 62 , 63 , 64 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 75 , 78 , 79 , 80 , 81 , 82 ]; and LEC EV markers [ 76 ]. ACE, angiotensin converting enzyme; ADAM15/17, disintegrin and metalloproteinase domain-containing protein 15/17; ALIX, apoptosis-linked gene (ALG)-2 interacting protein X; ANGPTL2, angiopoieitin-like protein 2; ARF6, ADP-ribosylation factor 6; Casp3; caspase 3; CAV1, caveolin 1; CCL2/5, chemokine ligand 2/5; CD, cluster of differentiation; CX3CL1, fractalkine; EDF-1, endothelial differentiation-related factor-1; EGFR, epidermal growth factor receptor; eIFs, eukaryotic initiation factors; FABP4, fatty acid binding protein 4; FLOT-1/2, flotillin 1/2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GMFγ, glia maturation factor gamma; HSP70/90, heat-shock protein 70/90; HSPß1, heat-shock protein ß1; IR, insulin receptor; ITGBL1, integrin subunit beta-like 1; LDL, low-density lipoprotein; (l)ncRNA, (long) non-coding RNA; LOXL2, lysyl oxidase-like 2; LPAR3, lysophosphatidic acid receptor 3; LRPs, low-density lipoprotein receptor-related proteins; MEK1/2, mitogen-activated protein kinase 1/2; MFGE8, milk fat globule-epidermal growth factor 8 protein (lactadherin); miR, micro RNA; MMP-2/-9, matrix metalloproteinase-2/-9; MT1-MMP, membrane-type 1 matrix metalloproteinase; NRP-1/-2, neuropilin-1/-2; PGF, placental growth factor; PKN, protein kinases; PS, phosphatidylserine; PTX3, pentraxin-related protein; RAB/RAB11, Ras superfamily of small G proteins (GTPases); ROK, Roh-associated kinases; S1PR1/3, sphingosine-1-phosph...…”

Exosomes in the Tumor Microenvironment: From Biology to Clinical Applications

“…Despite the benefits of 3D cultures, to date, few works have studied the role of immobilized exosomes in the extracellular matrix of the TME. However, bioprinting technology has allowed the evaluation of the exosome effects on extracellular matrix remodeling [101,[292][293][294]. This is because bioprinting technology is a powerful tool employed for tissue engineering, which allows for the precise placement of cells, biomaterials, and biomolecules in spatially predefined locales within confined 3D structures [295].…”