Nitrate-Functionalized poly(ε-Caprolactone) Small-Diameter Vascular Grafts Enhance Vascular Regeneration via Sustained Release of Nitric Oxide

Artificial small-diameter vascular grafts (SDVG) fabricated from synthetic biodegradable polymers, such as poly(ε-caprolactone) (PCL), exhibit beneficial mechanical properties but are often faced with issues impacting their long-term graft success. Nitric oxide (NO) is an important physiological gasotransmitter with multiple roles in orchestrating vascular tissue function and regeneration. We fabricated a functional vascular graft by electrospinning of nitrate-functionalized poly(ε-caprolactone) that could release NO in a sustained manner via stepwise biotransformation in vivo. Nitrate-functionalized SDVG (PCL/NO) maintained patency following abdominal arterial replacement in rats. PCL/NO promoted cell infiltration at 3-months post-transplantation. In contrast, unmodified PCL SDVG showed slow cell in-growth and increased incidence of neointima formation. PCL/NO demonstrated improved endothelial cell (EC) alignment and luminal coverage, and more defined vascular smooth muscle cell (VSMC) layer, compared to unmodified PCL SDVG. In addition, release of NO stimulated Sca-1+ vascular progenitor cells (VPCs) to differentiate and contribute to rapid luminal endothelialization. Furthermore, PCL/NO inhibited the differentiation of VPCs into osteopontin-positive cells, thereby preventing vascular calcification. Overall, PCL/NO demonstrated enhanced cell ingrowth, EC monolayer formation and VSMC layer regeneration; whilst inhibiting calcified plaque formation. Our results suggested that PCL/NO could serve as promising candidates for improved and long-term success of SDVG implants.

Improved Repopulation Efficacy of Decellularized Small Diameter Vascular Grafts Utilizing the Cord Blood Platelet Lysate

Background: The development of functional bioengineered small-diameter vascular grafts (SDVGs), represents a major challenge of tissue engineering. This study aimed to evaluate the repopulation efficacy of biological vessels, utilizing the cord blood platelet lysate (CBPL). Methods: Human umbilical arteries (hUAs, n = 10) were submitted to decellularization. Then, an evaluation of decellularized hUAs, involving histological, biochemical and biomechanical analysis, was performed. Wharton’s Jelly (WJ) Mesenchymal Stromal Cells (MSCs) were isolated and characterized for their properties. Then, WJ-MSCs (1.5 × 106 cells) were seeded on decellularized hUAs (n = 5) and cultivated with (Group A) or without the presence of the CBPL, (Group B) for 30 days. Histological analysis involving immunohistochemistry (against Ki67, for determination of cell proliferation) and indirect immunofluorescence (against activated MAP kinase, additional marker for cell growth and proliferation) was performed. Results: The decellularized hUAs retained their initial vessel’s properties, in terms of key-specific proteins, the biochemical and biomechanical characteristics were preserved. The evaluation of the repopulation process indicated a more uniform distribution of WJ-MSCs in group A compared to group B. The repopulated vascular grafts of group B were characterized by greater Ki67 and MAP kinase expression compared to group A. Conclusion: The results of this study indicated that the CBPL may improve the repopulation efficacy, thus bringing the biological SDVGs one step closer to clinical application.

Comparative Evaluation on Impacts of Fibronectin, Heparin–Chitosan, and Albumin Coating of Bacterial Nanocellulose Small-Diameter Vascular Grafts on Endothelialization In Vitro

In this study, we contrast the impacts of surface coating bacterial nanocellulose small-diameter vascular grafts (BNC-SDVGs) with human albumin, fibronectin, or heparin–chitosan upon endothelialization with human saphenous vein endothelial cells (VEC) or endothelial progenitor cells (EPC) in vitro. In one scenario, coated grafts were cut into 2D circular patches for static colonization of a defined inner surface area; in another scenario, they were mounted on a customized bioreactor and subsequently perfused for cell seeding. We evaluated the colonization by emerging metabolic activity and the preservation of endothelial functionality by water soluble tetrazolium salts (WST-1), acetylated low-density lipoprotein (AcLDL) uptake assays, and immune fluorescence staining. Uncoated BNC scaffolds served as controls. The fibronectin coating significantly promoted adhesion and growth of VECs and EPCs, while albumin only promoted adhesion of VECs, but here, the cells were functionally impaired as indicated by missing AcLDL uptake. The heparin–chitosan coating led to significantly improved adhesion of EPCs, but not VECs. In summary, both fibronectin and heparin–chitosan coatings could beneficially impact the endothelialization of BNC-SDVGs and might therefore represent promising approaches to help improve the longevity and reduce the thrombogenicity of BNC-SDVGs in the future.

Fabrication of Highly Oriented Cylindrical Polyacrylonitrile, Poly(lactide-co-glycolide), Polycaprolactone and Poly(vinyl acetate) Nanofibers for Vascular Graft Applications

Small-diameter vascular grafts fabricated from synthetic polymers have found limited applications so far in vascular surgeries, owing to their poor mechanical properties. In this study, cylindrical nanofibrous structures of highly oriented nanofibers made from polyacrylonitrile, poly (lactide-co-glycolide) (PLGA), polycaprolactone (PCL) and poly(vinyl acetate) (PVAc) were investigated. Cylindrical collectors with alternate conductive and non-conductive segments were used to obtain highly oriented nanofibrous structures at the same time with better mechanical properties. The surface morphology (orientation), mechanical properties and suture retention of the nanofibrous structures were characterized using SEM, mechanical tester and universal testing machine, respectively. The PLGA nanofibrous cylindrical structure exhibited excellent properties (tensile strength of 9.1 ± 0.6 MPa, suture retention strength of 27N and burst pressure of 350 ± 50 mmHg) when compared to other polymers. Moreover, the PLGA grafts showed good porosity and elongation values, that could be potentially used for vascular graft applications. The combination of PLGA nanofibers with extracellular vesicles (EVs) will be explored as a potential vascular graft in future.

Near-Field Electrospinning and Characterization of Biodegradable Small Diameter Vascular Grafts

The ideal “off the shelf” tissue engineering, small-diameter (< 6 mm inner diameter (ID)) vascular graft hinges on designing a template that facilitates transmural ingrowth of capillaries to regenerate an endothelized neointimal surface. Previous traditionally electrospun (TES) approaches to create bioresorbable vascular grafts lack the pore sizes required to facilitate transmural capillary ingrowth required for successful in situ neovascular regeneration. Therefore, the ability to create scaffolds with program-specific architectures independent of fiber diameter via the relatively recent sub-technique of near-field electrospinning (NFES) represents a promising solution to create tissue engineering vascular grafts. These programmed large pore sizes are anticipated to promote in situ regeneration and improve the outcomes as well as the quality of life of patients with arterial disease. In this dissertation, we manufactured via NFES as well as characterized biodegradable polydioxanone (PDO) small-diameter vascular grafts. Chapter 1 introduces the need for off-the-shelf, small-diameter vascular grafts to facilitate in situ regeneration, the process and pore size limitations of TES vascular grafts, and the promising use of NFES to develop precisely tailored PDO vascular grafts. Chapter 2 describes the process of NFES and details the current progress in NFES of biomedical polymers as well as the major limitations that exist in the field. Chapters 3, 4, and 5 contain primary research exploring the creation of an NFES vascular graft scaffold and characterizing the mechanical as well as biological response of these scaffolds. Specifically, in Chapter 3 we demonstrate a NFES apparatus designed around a commercial 3D printer to write PDO microfibers. The processing parameters of air gap, polymer concentration, translational velocity, needle gauge, and applied voltage were characterized for their effects on PDO fiber diameter. The processing parameters of polymer concentration and translational fiber deposition velocity were further characterized for their effects on fiber crystallinity and individual fiber uniformity. The precision of fiber stacking via a 3D printer was qualitatively evaluated to inform the creation of 3D scaffolds to guide the alignment of human gingival fibroblasts. It was found that fiber diameters correlate positively with polymer concentration, applied voltage, and needle gauge and inversely correlate with translational velocity and air gap distance. Individual fiber diameter variability decreases, and crystallinity increases with increasing translational fiber deposition velocity. These data resulted in the creation of tailored PDO 3D scaffolds which guided the alignment of primary human fibroblast cells. Together, these results suggest that NFES of PDO can be scaled to create precise geometries with tailored fiber diameters for vascular graft scaffolds. In Chapter 4, we demonstrated a NFES device to semi-stably write PDO microfibers. The polymer spinneret was programmed to translate in a stacking grid pattern, which resulted in a scaffold with highly aligned grid fibers that were intercalated with low density, random fibers. As a consequence of this random switching process, increasing the grid dimensions resulted in both a lower density of fibers in the center of each grid in the scaffold as well as a lower density of “rebar-like” stacked fibers per unit area. These hybrid architecture scaffolds resulted in tailorable as well as greater surface pore sizes as given by scanning electron micrographs and effective object permeability as indicated by fluorescent microsphere filtration compared to TES scaffolds of the same fiber diameter. Furthermore, these programmable scaffolds resulted in tailorability in the characterized mechanical properties ultimate tensile strength, percent elongation, yield stress, yield elongation, and Young’s modulus independent of fiber diameter compared to the static TES scaffold characterization. Lastly, the innate immune response of neutrophil extracellular traps (NETs) was further attenuated on NFES scaffolds compared to TES scaffolds. These results suggest that this novel NFES scaffold architecture of PDO can be highly tailored as a function of programming for small diameter vascular graft scaffolds. In Chapter 5, we created two types of NFES PDO architectures, as small-diameter vascular graft scaffolds. The first architecture type consisted of a 200 x 200 µm and 500 x 500 µm grid geometry with random fiber infill produced from one set of processing parameters, while the second architecture consisted of aligned fibers written in a 45°/45° and 20°/70° offset from the long axis, both on a 4 mm diameter cylindrical mandrel. These vascular graft scaffolds were characterized for their effective object transit pore size, mechanical properties, and platelet-material interactions compared to TES scaffolds and Gore-Tex® vascular grafts. It was found that effective pore size, given by 9.9 and 97 µm microsphere filtration through the scaffold wall for NFES grafts, was significantly more permeable compared to TES grafts and Gore-Tex® vascular grafts. Furthermore, the characterized mechanical properties of ultimate tensile strength, percent elongation, suture retention, burst pressure, and Young’s modulus were all tailorable for NFES grafts, independent of fiber diameter, compared to TES graft characterization. Lastly, platelet adhesion was attenuated on large pore size NFES grafts compared to the TES grafts which approximated the low level of platelet adhesion measured on Gore-Tex® grafts, with all grafts showing minimal platelet activation given by P-selectin surface expression. Together, these results suggest a highly tailorable process for the creation of the next generation of small-diameter vascular grafts. Lastly, Chapter 6 expounds future considerations for continuing research in NFES technology, NFES for general tissue engineering, and NFES for vascular tissue engineering as well as gives final conclusions. Together, the finding of this dissertation indicated that NFES vascular grafts result in seamless, small diameter tubular scaffolds with programmable pore sizes on the magnitude anticipated to facilitate transmural endothelialization as well as programmable mechanical properties that approximate native values. Thus, this work represents the next step in developing bioinstructive designed scaffolds to facilitate in situ vascular regeneration to improve the outcomes as well as the quality of life of patients with arterial vascular disease.