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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.

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.

Objective Elastogenesis within the medial layer of the aortic wall involves a cascade of events orchestrated primarily by smooth muscle cells, including transcription of elastin and a cadre of elastin chaperone matricellular proteins, deposition and cross-linking of tropoelastin coacervates, and maturation of extracellular matrix fiber structures to form mechanically competent vascular tissue. Elastic fiber disruption is associated with aortic aneurysm; in aneurysmal disease a thin and weakened wall leads to a high risk of rupture if left untreated, and non-surgical treatments for small aortic aneurysms are currently limited. This study analyzed the effect of adipose-derived stromal cell secreted factors on each step of the smooth muscle cell elastogenesis cascade within a three-dimensional fibrin gel culture platform. Approach and results We demonstrate that adipose-derived stromal cell secreted factors induce an increase in smooth muscle cell transcription of tropoelastin, fibrillin-1, and chaperone proteins fibulin-5, lysyl oxidase, and lysyl oxidase-like 1, formation of extracellular elastic fibers, insoluble elastin and collagen protein fractions in dynamically-active 30-day constructs, and a mechanically competent matrix after 30 days in culture. Conclusion Our results reveal a potential avenue for an elastin-targeted small aortic aneurysm therapeutic, acting as a supplement to the currently employed passive monitoring strategy. Additionally, the elastogenesis analysis workflow explored here could guide future mechanistic studies of elastin formation, which in turn could lead to new non-surgical treatment strategies., Highlights • Stromal cells stimulate smooth muscle cells (SMC) using paracrine signals. • Stimulated SMC make RNA for both elastin and associated proteins. • After protein synthesis, new elastic fibers form that contain insoluble elastin. • Stromal cell products could promote elastin production in vivo.

Macromolecular components of the vascular extracellular matrix (ECM), particularly elastic fibers and collagen fibers, are critical for the proper physiological function of arteries. When the unique biomechanical combination of these fibers is disrupted, or in the ultimate extreme where fibers are completely lost, arterial disease can emerge. Bioengineers in the realms of vascular tissue engineering and regenerative medicine must therefore ideally consider how to create tissue engineered vascular grafts containing the right balance of these fibers and how to develop regenerative treatments for situations such as an aneurysm where fibers have been lost. Previous work has demonstrated that the primary cells responsible for vascular ECM production during development, arterial smooth muscle cells (SMCs), can be induced to make new elastic fibers when exposed to secreted factors from adipose-derived stromal cells. To further dissect how this signal is transmitted, in this study, the factors were partitioned into extracellular vesicle (EV)-rich and EV-depleted fractions as well as unseparated controls. EVs were validated using electron microscopy, dynamic light scattering, and protein quantification before testing for biological effects on SMCs. In 2D culture, EVs promoted SMC proliferation and migration. After 30 days of 3D fibrin construct culture, EVs promoted SMC transcription of the elastic microfibril gene FBN1 as well as SMC deposition of insoluble elastin and collagen. Uniaxial biomechanical properties of strand fibrin constructs were no different after 30 days of EV treatment versus controls. In summary, it is apparent that some of the positive effects of adipose-derived stromal cells on SMC elastogenesis are mediated by EVs, indicating a potential use for these EVs in a regenerative therapy to restore the biomechanical function of vascular ECM in arterial disease.

Objective Tissue-engineered vascular grafts containing adipose-derived mesenchymal stem cells offer an alternative to small-diameter vascular grafts currently used in cardiac and lower-extremity revascularization procedures. Adipose-derived, mesenchymal stem cell–infused, tissue-engineered vascular grafts have been shown to promote remodeling and vascular homeostasis in vivo and offer a possible treatment solution for those with cardiovascular disease. Unfortunately, the time needed to cultivate adipose-derived mesenchymal stem cells remains a large hurdle for tissue-engineered vascular grafts as a treatment option. The purpose of this study was to determine if stromal vascular fraction (known to contain progenitor cells) seeded tissue-engineered vascular grafts would remain patent in vivo and remodel, allowing for a “same-day” process for tissue-engineered vascular graft fabrication and implantation. Methods Stromal vascular fraction, obtained from adult human adipose tissue, was seeded within 4 hours after acquisition from the patient onto poly(ester urethane)urea bilayered scaffolds using a customized rotational vacuum seeding device. Constructs were then surgically implanted as abdominal aortic interposition grafts in Lewis rats. Results Findings revealed patency in 5 of 7 implanted scaffolds at 8 weeks, along with neotissue formation and remodeling occurring in patent tissue-engineered vascular grafts. Patency was documented using angiography and gross inspection, and remodeling and vascular components were detected using immunofluorescent chemistry. Conclusions A “same-day” cell-seeded, tissue-engineered vascular graft can remain patent after implantation in vivo, with neotissue formation and remodeling occurring by 8 weeks.