A major proportion of cardiovascular diseases is originated from the occlusion of vital blood vessels carrying oxygenated blood to the target organ. Aberrant blood flow leads to ischemia and organ failure. One of the most predominant examples is the occlusion of the coronary artery causing myocardial infarction and eventual heart failure. Balloon angioplasty, laser angioplasty, and placement of stents are some of the effective clinical options but concomitantly are associated with secondary complications requiring follow-up surgical interventions. Autologous bypass grafting remains the gold standard for the past six decades but faces several limitations, including a shortage of healthy donor vessels. Small diameter (<6mm) blood vessels pose further challenges owing to their high failure rates. Tissue-engineered vascular grafts are currently the best-suited option, wherein a bioresorbable biomaterial is used to harness the body’s regeneration capability.
While synthetic biomaterials release toxic byproducts upon degradation, the implementation of natural polymers is promising in regeneration.
A substantial research impetus in the field is now focused on using natural ECM proteins as building blocks to manufacture TEVGs. In this regard, some of the prevalently explored natural polymers in tissue engineering are collagen, fibrin, elastin, gelatin, and silk fibroin. Collagen scaffolds are biologically active and predominantly contain cell-binding sites; however, the recapitulation of native-like fibril assembly conferring alpha-helix structure is a formidable challenge. As a result, collagen-based grafts succumb to physiological blood pressure owing to poor mechanical properties. Similarly, fibroblast embedded fibrin gel-based grafts require long- term maturation in a simulated dynamic environment. On the other hand, the fibrin gel provides a congenial structural framework to cells as they secrete their own ECM, providing a mechanically resilient implantable TEVG. Inadequate in situ elastin production and lamellar organization in implanted TEVGs is one of the persisting limiting factors. Incorporating either smooth muscle cells generated elastin or engineered synthetic elastin in vascular grafts before implantation is one prospective solution. While some of these natural biomaterials offer outstanding bioactivity, their questionable mechanical properties have limited direct implementation of such polymers.
Moreover, their large-scale production is time-intensive and expensive.
Silk is a natural, versatile protein biopolymer produced by various insects (silkworms, spiders, scorpions, mites, and flies). This ancient textile material has long been used as sutures in surgery owing to its biocompatibility and remarkable tensile strength. Considering an array of favorable inherent properties, the past few decades have witnessed silk use as a potential biomaterial for various tissue engineering applications. Among other variants, regenerated silk fibroin obtained from Bombyx mori silkworm cocoons is widely explored for regenerative medicine. Lately, a few of the silk-made bioengineered products were approved by FDA for clinical implementation, including SERI surgical scaffold® and Silk Voice®. Few characteristic properties that make silk a suitable choice for tissue engineering applications are: 1) biocompatibility, 2) tunable biodegradation, 3) minimally immunogenic, 4) ability to adapt various formats (3D scaffolds, thin films, nanofibers, microspheres, nanoparticles, hydrogels, etc.), 5) extraordinary mechanical strength, 6) easy accessibility, 7) cost-effective, 8) easy green processing. Without exception, silk biomaterials are under active investigation to explore their potential for vascular tissue engineering applications. Notwithstanding the remarkable promise of silk in various regenerative medicine fields, its involvement in creating tissue-engineered vascular reconstruction products has witnessed a comparative scarcity. Moreover, a significant proportion of prior literature suggests the predominant application of silk fibroin obtained from domesticated Bombyx mori silk variety, possibly due to the geographically restricted distribution of many silk types. Nevertheless, lately, researchers are actively investigating silk-based TEVGs prepared by diverse fabrication methodologies in pre-clinical settings and have shown very encouraging outcomes.
We first identified various parameters, which are of utmost importance for the clinical translation of TEVGs. The anti-thrombogenic property of biomaterial is crucial to allow blood flow through the scaffold lumen without coagulation. The fabricated graft should sustain the physiological blood pressure without failure while concomitantly allowing the formation of neo- tissue. A controlled innate immune response facilitates graft remodeling. From a clinical perspective, readily available or fast fabricating grafts would be suitable. In addition, a cell-free graft would have a better chance of regulatory clearance. Affordability is another essential aspect for the broader applicability of tissue-engineered grafts. In this thesis work, we have explored the potential of mulberry (Bombyx mori) and Indian endemic non-mulberry silk (Antheraea assama) for vascular tissue engineering applications. The additional advantageous aspect of non-mulberry
silk is their inherent presence of RGD (Arg-Gly-Asp) cell binding tripeptide, which is envisaged to facilitate cell adhesion and growth. Moreover, poly-alanine repeats in the heavy chain of non- mulberry silk render remarkable mechanical strength, which remains one of the vital prerequisites while developing vascular grafts.
Considering the enormous scope of silk biomaterials, we have used various progressive strategies to fabricate small-diameter vascular grafts. We first show the favorable cell-material interaction and implications of surface patterns to induce native-like unidirectional alignment resulting in functional improvement. We further report a novel facile methodology to develop bi- layered biomimetic grafts, which were investigated either in cell-seeded format (adipose stem cells) or cell-free platform (functionalized with human Wharton’s jelly). We have shown the superior performance of non-mulberry silk-based TEVGs in rat and rabbit implantation models through various defined objectives. Finally, we provide a TEVG platform having the potential to locally deliver the desired bioactive cargo towards improving clinically viable acellular grafts.
These grafts could be fabricated in a highly reproducible manner and are potential candidates for ready availability. Different hypothetical strategies are analyzed through the following defined objectives:
1. Combinatorial analysis of silk films’ innate physicochemical properties and surface topography on the functional behavior of vascular cells.
2. Development of small-diameter vascular grafts using patterned silk films recapitulating native arterial structure.
3. Development of bi-layered tubular silk scaffolds consisting of inner porous freeze-dried layer coated with outer dense electrospun layer and their functional analysis in a rat aortic interposition model as cell-seeded vascular grafts.
4. Acellular silk-decellularized human Wharton’s jelly extracellular matrix composite tubular scaffolds and their functional analysis in rabbit jugular vein as interposition graft for vascular tissue engineering applications.
5. Acellular silk lyogel conduits impregnated with bioactive polymeric microparticles as potential substitutes for vascular tissue engineering applications.