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.
In situ tissue regeneration using a novel tissue-engineered, small-caliber vascular graft without cell seeding
