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Cardiovascular disease is the leading cause of death worldwide, often associated with coronary artery occlusion. A common intervention for arterial blockage utilizes a vascular graft to bypass the diseased artery and restore downstream blood flow; however, current clinical options exhibit high long-term failure rates. Our goal was to develop an off-the-shelf tissue-engineered vascular graft capable of delivering a biological payload based on the monocyte recruitment factor C-C motif chemokine ligand 2 (CCL2) to induce remodeling. Bi-layered silk scaffolds consisting of an inner porous and outer electrospun layer were fabricated using a custom blend of Antherea Assama and Bombyx Mori silk (lyogel). Lyogel silk scaffolds alone (LG), and lyogel silk scaffolds containing microparticles (LGMP) were tested. The microparticles (MPs) were loaded with either CCL2 (LGMP+) or water (LGMP-). Scaffolds were implanted as abdominal aortic interposition grafts in Lewis rats for 1 and 8 weeks. 1-week implants exhibited patency rates of 50% (7/14), 100% (10/10), and 100% (5/5) in the LGMP-, LGMP+, and LG groups, respectively. The significantly higher patency rate for the LGMP+ group compared to the LGMP- group (p = 0.0188) suggests that CCL2 can prevent acute occlusion. Immunostaining of the explants revealed a significantly higher density of macrophages (CD68+ cells) within the outer vs. inner layer of LGMP- and LGMP+ constructs but not in LG constructs. After 8 weeks, there were no significant differences in patency rates between groups. All patent scaffolds at 8 weeks showed signs of remodeling; however, stenosis was observed within the majority of explants. This study demonstrated the successful fabrication of a custom blended silk scaffold functionalized with cell-mimicking microparticles to facilitate controlled delivery of a biological payload improving their in vivo performance. Statement of significance This study outlines the development of a custom blended silk-based tissue–engineered vascular graft (TEVG) for use in arterial bypass or replacement surgery. A custom mixture of silk was formulated to improve biocompatibility and cellular binding to the tubular scaffold. Many current approaches to TEVGs include cells that encourage graft cellularization and remodeling; however, our technology incorporates a microparticle based delivery platform capable of delivering bioactive molecules that can mimic the function of seeded cells. In this study, we load the TEVGs with microparticles containing a monocyte attractant and demonstrate improved performance in terms of unobstructed blood flow versus blank microparticles. The acellular nature of this technology potentially reduces risk, increases reproducibility, and results in a more cost-effective graft when compared to cell-based options.

Vascular tissue engineering is aimed at developing regenerative vascular grafts to restore tissue function by bypassing or replacing defective arterial segments with tubular biodegradable scaffolds. Scaffolds are often combined with stem or progenitor cells to prevent acute thrombosis and initiate scaffold remodeling. However, there are limitations to cell-based technologies regarding safety and clinical translation. Extracellular vesicles (EVs) are nanosized particles released by most cell types, including stem and progenitor cells, that serve to transmit protein and RNA cargo to target cells throughout the body. EVs have been shown to replicate the therapeutic effect of their parent cells; therefore, EVs derived from stem or progenitor cells may serve as a more translatable, cell-free, therapeutic base for vascular scaffolds. Our study aims to determine if EV incorporation provides a positive effect on graft patency and remodeling

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.

This study addresses a crucial gap in the literature by characterising the relationship between urethral tissue mechanics, composition and gross structure. We then utilise these data to develop a biomimetic urethral scaffold with physical properties that more accurately mimic the native tissue than existing gold standard scaffolds; small intestinal submucosa (SIS) and urinary bladder matrix (UBM). Nine human urethra samples were mechanically characterised using pressure-diameter and uniaxial extension testing. The composition and gross structure of the tissue was determined using immunohistological staining. A pressure stiffening response is observed during the application of intraluminal pressure. The elastic and viscous tissue responses to extension are free of regional or directional variance. The elastin and collagen content of the tissue correlates significantly with tissue mechanics. Building on these data, a biomimetic urethral scaffold was fabricated from collagen and elastin in a ratio that mimics the composition of the native tissue. The resultant scaffold is comprised of a dense inner layer and a porous outer layer that structurally mimic the submucosa and corpus spongiosum layers of the native tissue, respectively. The porous outer layer facilitated more uniform cell infiltration relative to SIS and UBM when implanted subcutaneously (p The tissue characterisation data presented herein paves the way for the development of biomimetic urethral grafts, and the novel scaffold we develop demonstrates positive findings that warrant furtherin vivoevaluation.

Vascular tissue engineering is a field of regenerative medicine that restores tissue function to defective sections of the vascular network by bypass or replacement with a tubular, engineered graft. The tissue engineered vascular graft (TEVG) is comprised of a biodegradable scaffold, often combined with cells to prevent acute thrombosis and initiate scaffold remodeling. Cells are most effectively incorporated into scaffolds using bulk seeding techniques. While our group has been successful in uniform, rapid, bulk cell seeding of scaffolds for TEVG testing in small animals using our well-validated rotational vacuum technology, this approach was not directly translatable to large scaffolds, such as those required for large animal testing or human implants. The objective of this study was to develop and validate a semi-automated cell seeding device that allows for uniform, rapid, bulk seeding of large scaffolds for the fabrication of TEVGs appropriately sized for testing in large animals and eventual translation to humans. Validation of our device revealed successful seeding of cells throughout the length of our tubular scaffolds with homogenous longitudinal and circumferential cell distribution. To demonstrate the utility of this device, we implanted a cell seeded scaffold as a carotid interposition graft in a sheep model for 10 weeks. Graft remodeling was demonstrated upon explant analysis using histological staining and mechanical characterization. We conclude from this work that our semi-automated, rotational vacuum seeding device can successfully seed porous tubular scaffolds suitable for implantation in large animals and provides a platform that can be readily adapted for eventual human use.