Vascular Damage and Repair - Are Small-Diameter Vascular Grafts Still the "Holy Grail" of Tissue Engineering?

Abstract

Cardiovascular diseases are the most important cause of morbidity and mortality in the civilized world. Stenosis or occlusion of blood vessels leads not only to events that are directly life-threatening, such as myocardial infarction or stroke, but also to a significant reduction in quality of life, for example in lower limb ischemia as a consequence of metabolic diseases. The first synthetic polymeric vascular replacements were used clinically in the early 1950s. However, they proved to be suitable only for larger-diameter vessels, where the blood flow prevents the attachment of platelets, pro-inflammatory cells and smooth muscle cells on their inner surface, whereas in smaller-diameter grafts (6 mm or less), these phenomena lead to stenosis and failure of the graft. Moreover, these polymeric vascular replacements, like biological grafts (decellularized or devitalized), are cell-free, i.e. there are no reconstructed physiological layers of the blood vessel wall, i.e. an inner layer of endothelial cells to prevent thrombosis, a middle layer of smooth muscle cells to perform the contractile function, and an outer layer to provide innervation and vascularization of the vessel wall. Vascular substitutes with these cellular components can be constructed by tissue engineering methods. However, it has to be admitted that even about 70 years after the first polymeric vascular prostheses were implanted into human patients, there are still no functional small-diameter vascular grafts on the market. The damage to small-diameter blood vessels has to be addressed by endovascular approaches or by autologous vascular substitutes, which leads to some skepticism about the potential of tissue engineering. However, new possibilities of this approach lie in the use of modern technologies such as 3D bioprinting and/or electrospinning in combination with stem cells and pre-vascularization of tissue-engineered vascular grafts. In this endeavor, sex-related differences in the removal of degradable biomaterials by the cells and in the behavior of stem cells and pre-differentiated vascular cells need to be taken into account. Key words: Blood vessel prosthesis, Regenerative medicine, Stem cells, Footprint-free iPSCs, sr-RNA, Dynamic bioreactor, Sex-related differences.

Fig. 1

Clinical achievements in tissue engineering of vascular grafts. Created with BioRender.com

Fig. 2

Immunohistological staining of von Willebrand factor (A, C) and calponin (B, D) of endothelialized porcine carotid artery that had been decellularized and seeded with human adipose tissue-derived stem cells (A, B) or human bone marrow-derived stem cells (C, D) for 14 days and human umbilical vein endothelial cells for two days of in vitro culture. Hematoxylin-eosin staining of control native (E) and decellularized arteries, tile scan (F). A–E: Olympus IX 71 epifluorescence microscope, DP80 digital camera; A–D: obj.×4, scale bar = 500 μm; E: obj.×10, scale bar = 200 μm; F: ZEISS Axio Scan. Z1 Slide Scanner, obj.×20.

Fig. 3

The influence of the cultivation substrate on the differentiation of ADSCs towards VSMCs. The correlation of relative mRNA expression of genes for calponin 1 (CNN1) and smoothelin (SMTN) in human adipose-derived stem cells cultivated in differentiation medium on standard tissue culture polystyrene (PS), PS coated with type 1 collagen (PS + Col) or with fibronectin (PS + FN). Measured by real-time PCR. Arithmetic mean ± SD from 2–3 samples for each experimental group and time interval. ANOVA, Student-Newman-Keuls method. Statistical significance: * p≤0.05 in comparison with corresponding control PS samples.

Fig. 4

A: Capillary-like network formation in a collagen hydrogel with embedded HUVECs and ADSCs immigrating from an underlying fibrin-modified electrospun nanofibrous polylactide membrane after 14 days in culture. B: Immunofluorescence staining of β-actin (red) and calponin (green) in human ADSCs in collagen gels on day 14 of culture in EGM-2 medium. C: A monolayer of HUVECs originating from the openings (arrows) of capillary-like structures on the surface of the collagen hydrogel. A, C: Both cell types were stained with phalloidin for cytoskeletal F-actin (conjugated with Atto 488; all images; red), and with DAPI for cell nuclei (all images; blue). The CD31 membrane marker of HUVECs was visualized by immunofluorescence (Alexa 633; turquoise). D, E: bottom and side view of the construct, where the collagen hydrogel was enriched with fibronectin (10 μg/ml). Both HUVECs and ADSCs were stained with phalloidin for the cytoskeletal F-actin (red), and with DAPI for the cell nuclei (blue). Von Willebrand factor, a marker of HUVECs, was visualized by immunofluorescence (green). Scale bar 50 μm. A, C, D, E: Dragonfly 503 spinning disk confocal microscope, obj. ×20 (A, D, E), obj. ×40 (C). B: Zeiss Z.1 light-sheet microscope, obj. ×10 for excitation, obj. ×20 for detection, zoom ×0.4 tile scans.

Fig. 5

Performance of a PET vascular prosthesis modified with fibrin and fibronectin, seeded with autologous endothelial cells and implanted into pigs. A: the inner surface pre-seeded with endothelial cells in vitro prior to the implantation; B: the gross morphology of explanted prostheses on day 22 after implantation: left – non-modified, right – pre-endothelialized; C, D, E: proximal, middle and distal parts of the prosthesis, respectively; F: control co-culture of endothelial cells and VSMCs on a glass coverslip. Immunofluorescence stain of von Willebrand factor, a marker of endothelial cells (red) and α-actin, a marker of VSMCs (green). The cell nuclei are counterstained with Hoechst 33342 (blue). Confocal microscope Leica SP2.