The combination of a natural polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and a synthetic hydrophobic polymer poly(ε-caprolactone) (PCL) is promising for the preparation of biodegradable and biocompatible small-diameter vascular grafts for bypass surgery. However, physico-mechanical properties and endothelialization rate of PHBV/PCL grafts are poor. We suggested that incorporation of vascular endothelial growth factor (VEGF) into PHBV/PCL grafts may improve their physico-mechanical properties and enhance endothelialization. Here we compared morphology, physico-mechanical properties, and in vivo performance of electrospun small-diameter vascular grafts prepared from PHBV/PCL with and without VEGF. Structure of the graft surface and physico-mechanical properties were examined by scanning electron microscopy and universal testing machine, respectively. Grafts were implanted into rat abdominal aorta for 1, 3, and 6 months with the further histological, immunohistochemical, and immunofluorescence examination. PHBV/PCL grafts with and without VEGF were highly porous and consisted mostly of nanoscale and microscale fibers, respectively. Mean pore diameter and mean pore area were significantly lower in PHBV/PCL/VEGF compared to PHBV/PCL grafts (1.47 μm and 10.05 μm2; 2.63 μm and 47.13 μm2, respectively). Durability, elasticity, and stiffness of PHBV/PCL grafts with VEGF were more similar to internal mammary artery compared to those without, particularly 6 months postimplantation. Both qualitative examination and quantitative image analysis showed that three-fourths of PHBV/PCL grafts with VEGF were patent and had many CD31-, CD34-, and vWF-positive cells at their inner surface. However, all PHBV/PCL grafts without VEGF were occluded and had no or a few CD31-positive cells at the inner surface. Therefore, VEGF enhanced endothelialization and improved graft patency at all the time points in a rat abdominal aorta replacement model. In conclusion, PHBV/PCL grafts with VEGF have better biocompatibility and physico-mechanical properties compared to those without. Incorporation of VEGF improves graft patency and accelerates formation of endothelial cell monolayer.

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.

Small diameter arterial bypass grafts are considered as unmet clinical need since the current grafts have poor patency of 25% within 5 years. We have developed a 3D scaffold manufactured from natural and synthetic biodegradable polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(𝜀-caprolactone) (PCL), respectively. Further to improve the biophysical properties as well as endothelialisation, the grafts were covalently conjugated with arginine-glycine-aspartic acid (RGD) bioactive peptides. The biophysical properties as well as endothelialisation of PHBV/PCL and PCL 2 mm diameter bypass grafts were assessed with and without biofunctionalisation with RGD peptides in vitro and in vivo. Morphology of the grafts was assessed by scanning electron microscopy, whereas physico-mechanical properties were evaluated using a physiological circulating system equipped with a state of art ultrasound vascular wall tracking system. Endothelialisation of the grafts in vitro and in vivo were assessed using a cell viability assay and rat abdominal aorta replacement model, respectively. The biofunctionalisation with RGD bioactive peptides decreased mean fiber diameter and mean pore area in PHBV/PCL grafts; however, this was not the case for PCL grafts. Both PHBV/PCL and PCL grafts with RGD peptides had lower durability compared to those without; these durability values were similar to those of internal mammary artery. Modification of PHBV/PCL and PCL grafts with RGD peptides increased endothelial cell viability in vitro by a factor of eight and enhanced the formation of an endothelial cell monolayer in vivo 1 month postimplantation. In conclusion, PHBV/PCL small-caliber graft can be a suitable 3D scaffold for the development of a tissue engineering arterial bypass graft.

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.

25омплексные проблемы сердечно-сосудистых заболеваний К Highlights • The study demonstrates the benefits of using functionalized small-diameter biodegradable vascular graft for de novo vascular tissue formation.• The release of growth factors and chemoattractant molecules into the vascular bed after the graft implantation stimulates neotissue formation and levels out inflammation and calcification. BackgroundCurrently, the search for the bioactive molecules capable of promoting formation of the vascular tissue is still ongoing. We have previously demonstrated that incorporation of the growth factors and chemoattractant molecules into the biodegradable tubular scaffolds can increase their primary patency upon the implantation into rat abdominal aorta. However, further studies are required to investigate tissue remodeling using functionalized vascular grafts with the same diameter as a replaced native vessel. AimTo investigate the specific aspects of de novo vascular tissue formation and calcification employing rat abdominal aorta interposition model and vascular grafts with 1.5 mm diameter with incorporated vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and stromal cell-derived factor (SDF)-1α. MethodsTubular grafts with a diameter of 1.5 mm were blended of poly(3-hydroxybutyrateco-3-hydroxyvalerate) and poly(ε-caprolactone) (PHBV/PCL). Grafts without growth factors were fabricated using standard electrospinning technique whilst grafts with incorporated growth factors were prepared utilizing emulsion electrospinning. VEGF was incorporated into the inner third, whereas bFGF and SDF-1α were incorporated into the outer two-thirds of the graft. Grafts were implanted into the abdominal aortas of Wistar rats for 1, 3, 6, and 12 months following scanning electron microscopy along with histological and immunofluorescent examination. ResultsPrimary patency of the grafts with VEGF, bFGF, and SDF-1α reached 93% indicative of structural integrity of the vascular tissue. Neither signs of inflammation nor severe calcification was detected. ConclusionAs in 2 mm diameter vascular grafts, incorporation of bioactive factors into 1.5 mm diameter grafts increased their long-term primary patency and improved vascular tissue formation in comparison with non-modified grafts.

We have previously developed a polycaprolactone (PCL) vascular graft with incorporated vascular endothelial growth factor (VEGF). Functioning of the PCL/VEGF graft in rat circulatory system over 1, 3 and 6 months after implantation into abdominal aorta was tested. Graft patency and formation of vascular wall elements were assessed histologically and by immunofluorescence staining for von Willebrand factor, CD31, CD34, and collagens I and IV and DAPI staining. Local application of VEGF promoted endothelialization and improved patency of the graft. The wall of the PCL/VEGF graft underwent remodeling due to active cellular infiltration and the extracellular matrix deposition.

Tissue-engineered vascular grafts are widely tested as a promising substitute for both arterial bypass and replacement surgery. We previously demonstrated that incorporation of VEGF into electrospun tubular scaffolds from poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(ε-caprolactone) enhances formation of an endothelial cell monolayer. However, an overdose of VEGF can induce tumor-like vasculature; thereby, other bioactive factors are needed to support VEGF-driven endothelialization and successful recruitment of smooth muscle cells. Utilizing emulsion electrospinning, we fabricated one-layer vascular grafts with either VEGF, bFGF, or SDF-1α, and two-layer vascular grafts with VEGF incorporated into the inner layer and bFGF and SDF-1α incorporated into the outer layer with the following structural evaluation, tensile testing, and in vivo testing using a rat abdominal aorta replacement model. The latter graft prototype showed higher primary patency rate. We found that the two-layer structure improved surface topography and mechanical properties of the grafts. Further, the combination of bFGF, SDF-1α, and VEGF improved endothelialization compared with VEGF alone, while bFGF induced a rapid formation of a smooth muscle cell layer. Taken together, these findings show that the two-layer structure and incorporation of bFGF and SDF-1α into the vascular grafts in combination with VEGF provide a higher primary patency and therefore improved in vivo performance.

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.