Development of small-diameter vascular grafts using patterned silk films recapitulating native arterial structure
3.1 Introduction
Cardiovascular malfunctioning is one of the leading causes of death globally and India is now being called ‘the world capital of cardiovascular diseases’. It is pertinent to mention that coronary artery occlusion incidences are more prominent (~50% of total cardiovascular patients). A recent report from World Health Organization (WHO) states that 17.3 million people died of cardiovascular diseases in 2008 and this number is expected to reach 23.3 million by 2030 [1].
Current surgical treatment methods require an adequate supply of native or native like vascular constructs so as to replace the diseased vessels. Autologous vessels, including saphenous and umbilical veins and mammary arteries serve as ‘gold standard’ for coronary replacement but almost one-third of patients do not have their veins appropriate for grafting due to pre-existing vein stripping, vascular disease and prior vein harvesting [4, 279]. The associated surgical cost and significant morbidity rate pose additional limitations.
Considering these limitations, there exists an urgent need to find a suitable alternative.
Tissue engineering, in this regard could serve as important tool for preparation of readily available, functional and biocompatible vascular grafts through scaffold based biomimetic approach.
Synthetic materials like polyethylene terephthalate (Dacron) and expanded polytetrafluoroethylene (ePTFE) are apt choice for large diameter (>6mm) vascular grafts and have been implanted successfully in thoracic and abdominal aorta [189, 280]. On the contrary their use for small caliber vessels has shown to cause thrombosis, anastomotic intimal hyperplasia and subsequent occlusion (reduced patency) due to compliance mismatch and inflammation [281].
This necessitates the selection of a suitable biomaterial that could withstand the continuous shear and vascular wall stretches with minimal energy loss and supports the growth of vascular cells.
Among others, silk represents its potential candidature for vascular tissue engineering owing to its easy accessibility, ease in processing, morphology control with immense modification options, extraordinary mechanical properties with flexibility, tunable degradability and hemocompatibility [114]. Further, amenability for fabrication into various forms like film, fiber, gel, sponge and particles broadens its usefulness and opens new portal of applications [165].
It is important to know that blood vessel is a layer by layer assembly of vascular cells arranged in a unique fashion so as to sustain the shear forces induced via blood flow. Recent endeavors implementing the use of silk for vascular tissue engineering application demonstrated the fabrication of porous silk microtube using layer-by-layer deposition and gel spinning methods
[158, 281, 282]. Such grafts although provides control over porosity but major drawback with these grafts is randomly arranged cells and their inability to maintain functional cellular phenotype.
Electrospun nanofibrous silk tubes certainly improved the prior fabrication approach but maintenance of long term mechanical compliance and poor control over mechanical and degradation properties imposed further limitations [283]. Silk composite were then investigated for their supposedly improved properties. In this regard, tri layered vascular grafts composed of elastin, polycaprolactone, silk and collagen were developed to match the required mechanical properties [284]. Silk micro-tube encapsulating heparin have been used as carrier for vascular endothelial growth factor (VEGF) sustained release, with concomitant hemocompatibility and endothelialization, thereby reducing the chance of thrombosis [165, 285]. A recent study projected the use of hybrid protein polymers containing silk and human recombinant tropoelastin to provide better tissue elasticity and extensibility [286]. These ‘top down approaches’ are based on the use of spongy scaffold as a template for engineering tubular construct followed by cell seeding in order to recapitulate compact and organized tissue. However, limitations in terms of long term mechanical compliance can be easily comprehended in situations that demand stretchable tube like structure with millimeter range wall thickness. Developing native like cellular arrangement and maintaining tissue integrity and complexity still remains a challenge in the field of vascular tissue engineering. Further limitations include co-culture of vascular cells, remodeling capability, long reproducible time and associated high cost.
Scaffold free approaches later came into existence that allows mimicking native cellular alignment and tissue integrity with higher fidelity. Cell sheet engineering is a good example of aforesaid technique. More than a decade ago, L'Heureux et al. successfully demonstrated the fabrication of human blood vessel using cell sheet engineering [287]. They have shown the subsequent rolling of sheets of SMCs and fibroblast cells over an inert mandrel followed by endothelial cell seeding in the luminal surface. The concept is although completely bio-based where one can precisely control the cellular and extracellular matrix (ECM) alignment; the rolling of the highly delicate cell sheets itself is tedious. In order to implement the concept of cell sheet engineering for preparing vascular graft and making the rolling process more feasible, recently people have tried to combine the principle of cell sheet engineering with the electrospinning technique [288, 289]. In this approach, aligned electrospun mat is used as a platform for cell seeding and after getting a confluent cell sheet, mat is rolled over the mandrel. This combined
approach made the rolling process more facile but require cellular infiltration during the maturation process. Limited infiltration of cells in electrospun scaffolds due to lesser pore size and inadequate surface properties need additional measures for graft success [283, 290].
Several reports have attested the applicability of Bombyx mori silk for vascular tissue engineering applications however non-mulberry silk varieties like Philosamia ricini and Antheraea assama are quite unexplored [189, 281, 282]. We have previously reported that non- mulberry silk varieties (e. g., Antheraea mylitta) support better cell attachment and proliferation owing to intrinsic presence of RGD motif (cell binding site) [291]. Recently, presence of similar cell binding motif was reported in A. assama [292]. Additionally, unique molecular architecture (polyalanine repeats without any intervening amino acid) of A. assama silk is reported to improve its mechanical properties- a considerable aspect of vascular tissue engineering [239, 293]. Hence, we attempted to explore the suitability of Indian endemic non-mulberry silk varieties (PR and AA) for vascular tissue engineering application.
In the current endeavor, we project an alternative approach by combining the cell sheet engineering and patterned silk films to overcome the above mentioned limitations. A schematic representation of our methodology to fabricate small diameter vascular graft is shown in Figure 3.1. Several reports have attested the applicability of B. mori silk for various tissue engineering applications but other non-mulberry silk varieties like P. ricini and A. assama are quite unexplored.
In this study, we used the latter in comparison with B. mori silk. This approach would serve as a suitable cell sheet platform simultaneously inducing the functional contractile phenotype of SMCs (alignment induced phenotypic transition). Also, it may be envisaged that cellular platform (silk film) would make the rolling process more facile and would not require long term maturation in pulsatile bioreactor to obtain sufficient mechanical strength and functionality. This consecutive rolling assembly is expected to exactly mimic the native cellular alignment (EC aligned longitudinally along the blood flow direction, SMCs and fibroblasts in the concentric arrangement improving mechanical contractility).
Figure 3.1. (A) Schematic representing the fabrication of patterned silk films and (B) rolling process of vascular cell sheets to obtain tri-layered biomimicking tissue engineered small diameter blood vessel.