Bioresorbable silk grafts for small diameter vascular tissue engineering applications: In vitro and in vivo functional analysis

Abstract

The success of tissue-engineered vascular graft (TEVG) predominantly relies on the selection of a suitable biomaterial and graft design. Natural biopolymer silk has shown great promise for various tissue-engineering applications. This study is the first to investigate Indian endemic non-mulberry silk (Antheraea assama-AA) - which inherits naturally superior mechanical and biological traits (e.g., RGD motifs) compared to Bombyx mori-BM silk, for TEVG applications. We designed bi-layered biomimetic small diameter AA-BM silk TEVGs adopting a new fabrication methodology. The inner layer showed ideally sized (~40 µm) pores with interconnectivity to allow cellular infiltration, and an outer dense electrospun layer that confers mechanical resilience. Biodegradation of silk TEVGs into amino acids as resorbable byproducts corroborates their in vivo remodeling ability. Following our previous reports, we surgically implanted human adipose tissue-derived stromal vascular fraction (SVF) seeded silk TEVGs in Lewis rats as abdominal aortic interposition grafts for 8 weeks. Adequate suture retention strength (0.45 ± 0.1 N) without any blood seepage post-implantation substantiate the grafts' viability. AA silk-based TEVGs showed superior animal survival and graft patency compared to BM silk TEVGs. Histological analysis revealed neo-tissue formation, host cell infiltration and graft remodeling in terms of extracellular matrix turnover. Altogether, this study demonstrates promising aspects of AA silk TEVGs for vascular tissue engineering applications. STATEMENT OF SIGNIFICANCE: Clinical 'off the shelf' implementation of tissue-engineered vascular grafts (TEVGs) remains a challenge. Achieving optimal blood vessel regeneration requires the use of bioresorbable materials having suitable degradation rates while producing minimal or no toxic byproducts. Host cell recruitment and preventing acute thrombosis are other pre-requisites for successful graft remodeling. In this study, for the first time we explored the use of naturally derived Indian endemic non-mulberry Antheraea assama silk in combination with Bombyx mori silk for TEVG applications by adopting a new biomimetic approach. Our bi-layered silk TEVGs were optimally porous, mechanically resilient and biodegradable. In vivo implantation in rat aorta showed long-term patency and graft remodeling by host cell infiltration and extracellular matrix deposition corroborating their clinical feasibility.

Keywords: Biodegradation; Non-mulberry silk; Tissue engineering; Tissue remodeling; Vascular grafts.

Figure 1:

Schematic representation of fabrication methodology of bi-layered small diameter silk scaffolds. The inner porous layer is prepared by molding and lyophilization based approach followed by coating with an outer nanofibrous electrospun layer.

Figure 2.. Morphometric analysis of bi-layered silk scaffolds.

(A) Representative images of tubular silk scaffolds and SEM micrographs showing internal porous architecture (CS: cross-section and lumen). MicroCT analysis of tubular silk scaffolds representing (B) 3D scaffold models and (C) Distribution of pore size of inner porous layer.

Figure 3.. Uniaxial tensile testing of silk scaffolds.

Average stress-strain curves in longitudinal (Long) and circumferential (Circ) directions for (A) BMES, (B) BM, (C) BAES and (D) BA silk scaffolds. Comparison of scaffold average modulus in the low (E, G) and high (F, H) stretch regions in longitudinal (E, F) and circumferential (G, H) directions. (##P<0.01, n.s. = not significant)

Figure 4:. Mechanical properties of bi-layered silk scaffolds.

(A) Comparison of suture retention force and (B) Suture retention tension between the two silk scaffold variants. (C) Comparison of β stiffness and (D) Dynamic compliance of silk scaffolds at initial (T=0h) and final (T=7h) time points under the influence of physiologically relevant pulsatile flow. (E) Creep analysis of silk scaffolds after 7h physiologically relevant pulsatile flow. (F) Comparison of burst pressure of silk scaffolds. (n.s. = not significant)

Figure 5:. In vitro degradation of tubular bi-layered silk scaffolds in the presence of protease XIV.

(A) SEM micrographs showing the scaffold morphology and effect of protease treatment over time. The higher magnification images on the right represent the scaffold degradation pattern (pore formation in scaffold struts) after 15 days of treatment. (B) Quantification of scaffold diameter after 15 days. (C) Graph representing the degradation of silk scaffold (in terms of percentage mass loss) over time in the presence or absence of protease enzyme. (‘/PRT’ represents the presence of protease and ‘/PBS’ represents the absence of protease) (##P<0.01, n.s. = not significant)

Figure 6:. Seeding tubular silk scaffolds with SVF cells and viability analysis.

(A) Silk scaffolds were mounted into the rotational vacuum cell seeding device prior to cellular infusion. The graph on the right represents the recorded luminal pressure at the proximal end with time during infusion of cells. (B) SVF seeded scaffolds were exposed to 48 h dynamic culture in a spinner flask and scaffold cross-sections were stained with DAPI (blue) indicating cell nuclei. ImageJ was used to map the cell distribution along the scaffold wall (representative images on the right side of the panel, dashed black line represents the scaffold wall); Scale bar: 200 μm. (C) Graph representing viability and proliferation of SVF cells cultured on silk scaffolds over 15 days under in vitro conditions. (## P<0.01)

Figure 7:. In vivo implantation of silk scaffolds and graft patency.

(A) A representative image of silk graft after aortic interposition implantation in a rat. (B) Representative images showing the explanted silk grafts after 8 weeks. (C) Representative images showing gross morphology of silk explants’ cross-section post 1 week and 8 week time points. Black arrows are showing the presence of neo-tissue in the lumen of silk explants. (D) Quantitative data representing in vivo graft performance. (E) Representative images of recorded angiograms showing graft patency after 8 weeks. White arrows represent the location of graft (infra-renal and above iliac bifurcation).

Figure 8:. Analysis of host cell infiltration, lumen diameter and wall thickness of explants.

(A) Representative immunofluorescence images of the middle section of vascular explants at different time points showing the infiltration of host cells (αSMA and calponin: SMCs, vWF: ECs and CD68: macrophages). The lumen of the explants is labeled as ‘*’ and marked with white dotted line. (B) Quantification of host cell infiltration in silk grafts. Graph representing (C) presence of CD68+ cells in silk grafts, (D) lumen diameter and (E) Wall thickness of vascular explants. (#P<0.05, ##P<0.01, n.s. = not significant)

Figure 9.. Histological analysis of extracellular matrix (ECM) production and graft remodeling.

(A) Representative histological images of the middle section of explanted grafts stained with H&E (Hematoxylin and eosin) for cell infiltration, PCRO (Picrosirius Red) for collagen (Red) and VVG (Verhoeff van Gieson) for elastin (Black). Scaffold lumen is marked as ‘*’ (Scale bar: 500 μm). Quantitative analysis of (B) collagen (n=3) and (C) elastin (n=3) in silk grafts compared with rat aorta. (#P<0.05, ##P<0.01, n.s. = not significant)