Introduction and review of literature
1.1 Introduction
Alarming mortality rates due to cardiovascular diseases (CVDs) arising from blockage or narrowing of vital blood vessels are of serious concern. The global disease burden and subsequent deaths are projected to escalate exponentially, with an estimated 23.4 million deaths in 2030 [1, 2]. The majority of CVDs stem from narrowing or blockage of vital blood vessels, which results in interrupted blood supply to the target organ and eventual ischemia. Some of the most commonly occurring CVDs are peripheral artery disease, cerebrovascular disease, deep vein thrombosis, and coronary artery disease. Bypassing the blood supply through an autologous graft via surgical intervention is currently the clinicians' preferred treatment choice [3]. Following are some of the reasons why autologous grafts are presently the gold standard for bypass surgeries: 1) they are physiologically the natural analogs of the diseased blood vessel which do not evoke immune response after implantation, hence no chance of graft rejection; 2) their lumen is naturally lined with endothelial cells which prevent thrombosis and SMC proliferation; 3) graft compliance and mechanical properties closely match the recipient site which attenuates the chance of intimal hyperplasia; 4) being originated from the patient him(her)self, no regulatory clearance is required;
5) they are readily available and finally; 6) they can grow in size with patient’s age. Commonly used autografts are saphenous vein, radial artery, and mammary artery [3].
The primary setback of using autologous grafts is donor site morbidity. In several cases, their unavailability due to prior harvesting or diseased condition exacerbates the situation precluding the possibility of auto-grafting. In this scenario, another viable option for clinicians is prosthetics [4]. Synthetic grafts (e.g., Gore-Tex and Dacron) currently dominate the global vascular grafts market, with an estimated value of 2.01 billion USD in 2018. A substantial portion is contributed by endovascular stent-grafts, hemodialysis access grafts, and peripheral vascular grafts (source: www.grandviewresearch.com). Notably, the application of synthetic grafts is limited to larger blood vessel (>6 mm diameter) replacement, and they face daunting obstacles when implemented for high pressure, small diameter blood vessels (<6 mm diameter) due to thrombosis and stenosis [5]. Besides, they are prone to chronic foreign body reaction, calcification, and risk of infection. The reason behind the poor performance of synthetic grafts is compliance mismatch and their non-biological composition [6]. Researchers are venturing into the surface
modification of these grafts to prevent thrombosis [7]. Amidst all the success, if we look at the vascular graft market with a clinical perspective, limited innovative products are coming up with the ever-persistent inability of synthetic grafts for smaller vessel replacement.
In pursuit of finding a clinically feasible alternative for small-diameter vessel replacement, tissue-engineered vascular grafts (TEVGs) are currently a popular choice [3, 8]. The aim of TEVGs is to provide the structural framework for host cells to re-grow the functional tissue. The overall idea is to design a scaffold (a tubular scaffold in this context) using a biodegradable polymer, seeding patient-specific cells, maturation in a naturally simulated dynamic microenvironment, and implantation into the patient. With time, the graft polymer is degraded and replaced by a newly formed ECM in a precisely balanced manner preserving the structural integrity, leaving behind a native-like regenerated tissue [5]. These lab-made grafts have the potential to mimic the autografts properties with high fidelity. Research in the field is now profoundly focused on conferring autologous grafts' properties into TEVGs for their clinical translation. Vascular tissue engineering has grown remarkably in the past few decades since the seminal groundwork laid by Weinberg and Bell in 1986 [9]. Scientists have investigated a plethora of different approaches to creating a clinically feasible small-diameter blood vessel that has remarkably improved our understanding.
While various synthetic and natural biomaterials have been explored to create bioresorbable vascular grafts, silk renders several advantages in terms of biocompatible degradation products, abundant availability and affordability.
Silk is an ancient textile material that has long been used as sutures in surgery owing to its biocompatibility and remarkable tensile strength. This natural protein biopolymer is produced by various insects (silkworms, scorpions, spiders, and flies) [10]. SF biomaterials provide numerous favorable inherent properties, that has enabled their remarkable performance in the past few decades as a potential biomaterial for various tissue engineering applications [11-13]. Among other variants, regenerated silk fibroin obtained from Bombyx mori silkworm cocoons is widely explored for regenerative medicine. Some of the silk-made bioengineered products have been approved by FDA for clinical implementation, including SERI surgical scaffold® and Silk Voice® [13].Few exemplary characteristic properties of SF biomaterial that make it a suitable choice for tissue engineering applications include: 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 [11, 14]. In addition to the other potential areas of regenerative medicine, silk biomaterials are actively being investigated to explore their potential for vascular tissue engineering applications [15]. Despite the remarkable promising aspects of SF biomaterials in various domains of tissue engineering, their implementation in creating tissue-engineered vascular grafts 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 wide availability. Other non-mulberry silk varieties have sparsely been explored in this domain, possibly due to their geographical restrictions. Nevertheless, lately, researchers are actively investigating silk-based TEVGs prepared by diverse fabrication methodologies in pre-clinical settings, and have shown very encouraging outcomes.
In this thesis, we have progressively explored various approaches to fabricate clinically viable silk-based small-diameter vascular grafts. Towards this end, both mulberry (Bombyx mori) and non-mulberry (Antheraea assama and Philosmia ricini) silk varieties are explored in the domain of vascular tissue engineering. In addition, crucial factors were identified that would improve the clinical feasibility of vascular grafts. These factors rely on either biomaterial property (biocompatibility, immune compatibility, blood compatibility, biodegradation, mechanical properties, cost-effectiveness) or fabrication approach (fabrication time/ready availability). From a biomaterial perspective, silk fibroin remains a rational choice, whereas various fabrication approaches are pragmatically investigated. This thesis work first investigates the cell-biomaterial interaction to substantiate the suitability of silk for vascular regeneration. The first fabrication approach capitalizes on the principle of bionics, wherein a cellular multilayered TEVG is explored while recapitulating the native-like cellular arrangement. In the second approach, a novel bi- layered vascular graft is developed, and the fabrication time is further reduced from months to days by implementing human SVF cells (without culture) instead of vascular cells. The third and fourth approaches set out to provide a ready available vascular graft while completely bypassing the cell seeding step. In these cases, the bioactivity is preserved by means of functionalizing the acellular grafts with decellularized human Wharton’s jelly matrix and CCL2, which locally provide cell-secreted bioactive paracrine factors conferring constructive graft remodeling.
Research work carried out in this thesis offers the excellent pre-clinical success of developed vascular grafts in animal models (rats/rabbits).