Endothelial colony-forming cells (ECFC) are currently considered as a promising cell population for the pre-endothelialization or pre-vascularization of tissue-engineered constructs, including small-diameter biodegradable vascular grafts. However, the extent of heterogeneity between ECFC and mature vascular endothelial cells (EC) is unclear. Here, we performed a transcriptome-wide study to compare gene expression profiles of ECFC, human coronary artery endothelial cells (HCAEC), and human umbilical vein endothelial cells (HUVEC). Characterization of the abovementioned cell populations was carried out by immunophenotyping, tube formation assay, and evaluation of proliferation capability while global gene expression profiling was conducted by means of RNA-seq. ECFC were similar to HUVEC in terms of immunophenotype (CD31⁺vWF⁺KDR⁺CD146⁺CD34^-CD133^-CD45^-CD90^-) and tube formation activity yet had expectedly higher proliferative potential. HCAEC and HUVEC were generally similar to ECFC with regards to their global gene expression profile; nevertheless, ECFC overexpressed specific markers of all endothelial lineages (NRP2, NOTCH4, LYVE1), in particular lymphatic EC (LYVE1), and had upregulated extracellular matrix and basement membrane genes (COL1A1, COL1A2, COL4A1, COL4A2). Proteomic profiling for endothelial lineage markers and angiogenic molecules generally confirmed RNA-seq results, indicating ECFC as an intermediate population between HCAEC and HUVEC. Therefore, gene expression profile and behavior of ECFC suggest their potential to be applied for a pre-endothelialization of bioartificial vascular grafts, whereas in terms of endothelial hierarchy they differ from HCAEC and HUVEC, having a transitional phenotype.

The blend of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) has recently been considered promising for vascular tissue engineering. However, it was shown that PHBV/PCL grafts require biofunctionalization to achieve high primary patency rate. Here we compared immobilization of arginine–glycine–aspartic acid (RGD)-containing peptides and the incorporation of vascular endothelial growth factor (VEGF) as two widely established biofunctionalization approaches. Electrospun PHBV/PCL small-diameter grafts with either RGD peptides or VEGF, as well as unmodified grafts were implanted into rat abdominal aortas for 1, 3, 6, and 12 months following histological and immunofluorescence assessment. We detected CD31⁺/CD34⁺/vWF⁺ cells 1 and 3 months postimplantation at the luminal surface of PHBV/PCL/RGD and PHBV/PCL/VEGF, but not in unmodified grafts, with the further observation of CD31⁺CD34⁻vWF⁺ phenotype. These cells were considered as endothelial and produced a collagen-positive layer resembling a basement membrane. Detection of CD31⁺/CD34⁺ cells at the early stages with subsequent loss of CD34 indicated cell adhesion from the bloodstream. Therefore, either conjugation with RGD peptides or the incorporation of VEGF promoted the formation of a functional endothelial cell layer. Furthermore, both modifications increased primary patency rate three-fold. In conclusion, both of these biofunctionalization approaches can be considered as equally efficient for the modification of tissue-engineered vascular grafts.

Tissue-engineered vascular graft for the reconstruction of small arteries is still an unmet clinical need, despite the fact that a number of promising prototypes have entered preclinical development. Here we test Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)Poly(ε-caprolactone) 4-mm-diameter vascular grafts equipped with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and stromal cell-derived factor 1α (SDF-1α) and surface coated with heparin and iloprost (PHBV/PCL[VEGF-bFGF-SDF]^Hep/Ilo, n = 8) in a sheep carotid artery interposition model, using biostable vascular prostheses of expanded poly(tetrafluoroethylene) (ePTFE, n = 5) as a control. Primary patency of PHBV/PCL[VEGF-bFGF-SDF]^Hep/Ilo grafts was 62.5% (5/8) at 24 h postimplantation and 50% (4/8) at 18 months postimplantation, while all (5/5) ePTFE conduits were occluded within the 24 h after the surgery. At 18 months postimplantation, PHBV/PCL[VEGF-bFGF-SDF]^Hep/Ilo grafts were completely resorbed and replaced by the vascular tissue. Regenerated arteries displayed a hierarchical three-layer structure similar to the native blood vessels, being fully endothelialised, highly vascularised and populated by vascular smooth muscle cells and macrophages. The most (4/5, 80%) of the regenerated arteries were free of calcifications but suffered from the aneurysmatic dilation. Therefore, biodegradable PHBV/PCL[VEGF-bFGF-SDF]^Hep/Ilo grafts showed better short- and long-term results than bio-stable ePTFE analogues, although these scaffolds must be reinforced for the efficient prevention of aneurysms.

Modification with Arg-Gly-Asp (RGD) peptides is a promising approach to improve biocompatibility of small-calibre vascular grafts but it is unknown how different RGD sequence composition impacts graft performance. Here we manufactured 1.5 mm poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(ε-caprolactone) grafts modified by distinct linear or cyclic RGD peptides immobilized by short or long amine linker arms. Modified vascular prostheses were tested in vitro to assess their mechanical properties, hemocompatibility, thrombogenicity and endothelialisation. We also implanted these grafts into rat abdominal aortas with the following histological examination at 1 and 3 months to evaluate their primary patency, cellular composition and detect possible calcification. Our results demonstrated that all modes of RGD modification reduce ultimate tensile strength of the grafts. Modification of prostheses does not cause haemolysis upon the contact with modified grafts, yet all the RGD-treated grafts display a tendency to promote platelet aggregation in comparison with unmodified counterparts. In vivo findings identify that cyclic Arg-Gly-Asp-Phe-Lys peptide in combination with trioxa-1,13-tridecanediamine linker group substantially improve graft biocompatibility. To conclude, here we for the first time compared synthetic small-diameter vascular prostheses with different modes of RGD modification. We suggest our graft modification regimen as enhancing graft performance and thus recommend it for future use in tissue engineering.

Superior polymers represent a promising alternative to mechanical and biological materials commonly used for manufacturing artificial heart valves. The study is aimed at assessing poly(styrene-block-isobutylene-block-styrene) (SIBS) properties and comparing them with polytetrafluoroethylene (Gore-tex^TM, a reference sample). Surface topography of both materials was evaluated with scanning electron microscopy and atomic force microscopy. The mechanical properties were measured under uniaxial tension. The water contact angle was estimated to evaluate hydrophilicity/hydrophobicity of the study samples. Materials’ hemocompatibility was evaluated using cell lines (Ea.hy 926), donor blood, and in vivo. SIBS possess a regular surface relief. It is hydrophobic and has lower strength as compared to Gore-tex^TM (3.51 MPa vs. 13.2/23.8 MPa). SIBS and Gore-tex^TM have similar hemocompatibility (hemolysis, adhesion, and platelet aggregation). The subcutaneous rat implantation reports that SIBS has a lower tendency towards calcification (0.39 mg/g) compared with Gore-tex^TM (1.29 mg/g). SIBS is a highly hemocompatible material with a promising potential for manufacturing heart valve leaflets, but its mechanical properties require further improvements. The possible options include the reinforcement with nanofillers and introductions of new chains in its structure.

The development of novel biodegradable vascular grafts of a small diameter (<6 mm) is an unmet clinical need for patients requiring arterial replacement. Here we performed a pre-clinical study of new small-caliber biodegradable vascular grafts using a sheep model of carotid artery implantation. The 4 mm diameter vascular grafts were manufactured using a mix of polyhydroxybutyrate/valerate and polycaprolactone supplemented with growth factors VEGF, bFGF and SDF-1α (PHBV/PCL-GFmix) and additionally modified by a polymer hydrogel coating with incorporation of drugs heparin and iloprost (PHBV/PCL-GFmix^Hep/Ilo). Animals with carotid artery autograft implantation and those implanted with clinically used GORE-TEX^® grafts were used as control groups. We observed that 24 h following surgery, animals with carotid artery autograft implantation showed 87.5% patency, while all the PHBV/PCL-GFmix and GORE-TEX^® grafts displayed thrombosis. PHBV/PCL-GFmix^Hep/Ilo grafts demonstrated 62.5% patency 24 h following surgery and it had remained at 50% 1 year post-operation. All the PHBV/PCL grafts completely degraded less than 1 year following surgery and were replaced by de novo vasculature without evidence of calcification. On the other hand, GORE-TEX^® grafts displayed substantial amounts of calcium deposits throughout graft tissues. Thus, here we report a potential clinical usefulness of PHBV/PCL grafts upon their additional modification by growth factors and drugs to promote endothelialization and reduce thrombogenicity.