CCL2 loaded microparticles promote acute patency in silk-based vascular grafts implanted in rat aortae.

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.

Extracellular Vesicles Derived from Primary Adipose Stromal Cells Induce Elastin and Collagen Deposition by Smooth Muscle Cells within 3D Fibrin Gel Culture.

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.

Development of a Semi-Automated, Bulk Seeding Device for Large Animal Model Implantation of Tissue Engineered Vascular Grafts.

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.

Extracellular Vesicles Enhance the Remodeling of Cell-Free Silk Vascular Scaffolds in Rat Aortae.

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 in vivo. We first assessed the effect of human adipose-derived mesenchymal stem cell (hADMSC) EVs on vascular cells using in vitro bioassays. We then developed an EV-functionalized vascular graft by vacuum-seeding EVs into porous silk-based tubular scaffolds. These constructs were implanted as aortic interposition grafts in Lewis rats, and their remodeling capacity was compared to that observed for hADMSC-seeded and blank (non-seeded) controls. The EV group demonstrated improved patency (100%) compared to the hADMSC (56%) and blank controls (82%) following eight weeks in vivo. The EV group also produced significantly more elastin (126.46%) and collagen (44.59%) compared to the blank group, while the hADMSC group failed to produce significantly more elastin (57.64%) or collagen (11.21%) compared to the blank group. Qualitative staining of the explanted neo-tissue revealed improved endothelium formation, increased smooth muscle cell infiltration, and reduced macrophage numbers in the EV group compared to the controls, which aids in explaining this group's favorable pre-clinical outcomes.

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

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.

Functional Vascular Tissue Engineering Inspired by Matricellular Proteins.

Modern regenerative medicine, and tissue engineering specifically, has benefited from a greater appreciation of the native extracellular matrix (ECM). Fibronectin, collagen, and elastin have entered the tissue engineer's toolkit; however, as fully decellularized biomaterials have come to the forefront in vascular engineering it has become apparent that the ECM is comprised of more than just fibronectin, collagen, and elastin, and that cell-instructive molecules known as matricellular proteins are critical for desired outcomes. In brief, matricellular proteins are ECM constituents that contrast with the canonical structural proteins of the ECM in that their primary role is to interact with the cell. Of late, matricellular genes have been linked to diseases including connective tissue disorders, cardiovascular disease, and cancer. Despite the range of biological activities, this class of biomolecules has not been actively used in the field of regenerative medicine. The intent of this review is to bring matricellular proteins into wider use in the context of vascular tissue engineering. Matricellular proteins orchestrate the formation of new collagen and elastin fibers that have proper mechanical properties-these will be essential components for a fully biological small diameter tissue engineered vascular graft (TEVG). Matricellular proteins also regulate the initiation of thrombosis via fibrin deposition and platelet activation, and the clearance of thrombus when it is no longer needed-proper regulation of thrombosis will be critical for maintaining patency of a TEVG after implantation. Matricellular proteins regulate the adhesion, migration, and proliferation of endothelial cells-all are biological functions that will be critical for formation of a thrombus-resistant endothelium within a TEVG. Lastly, matricellular proteins regulate the adhesion, migration, proliferation, and activation of smooth muscle cells-proper control of these biological activities will be critical for a TEVG that recellularizes and resists neointimal formation/stenosis. We review all of these functions for matricellular proteins here, in addition to reviewing the few studies that have been performed at the intersection of matricellular protein biology and vascular tissue engineering.

Adipose-derived stromal cell secreted factors induce the elastogenesis cascade within 3D aortic smooth muscle cell constructs.

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.

Decorin mimic promotes endothelial cell health in endothelial monolayers and endothelial-smooth muscle co-cultures.

Non-specific cytotoxins, including paclitaxel and sirolimus analogues, currently utilized as anti-restenotic therapeutics, affect not only smooth muscle cells (SMCs) but also neighbouring vascular endothelial cells (ECs). These drugs inhibit the formation of an intact endothelium following vessel injury, thus emphasizing the critical need for new candidate therapeutics. Utilizing our in vitro models, including EC monolayers and both hyperplastic and quiescent EC-SMC co-cultures, we investigated the ability of DS-SILY20 , a decorin mimic, to promote EC health. DS-SILY20 increased EC proliferation and migration by 1.5- and 2-fold, respectively, which corresponded to increased phosphorylation of ERK-1/2. Interestingly, IL-6 secretion and the production of both E-selectin and P-selectin were reduced in the presence of 10 µm DS-SILY20 , even in the presence of the potent pro-inflammatory cytokine platelet-derived growth factor (PDGF). In hyperplastic and quiescent EC-SMC co-cultures, DS-SILY20 treatment reduced the secretion of IFNγ, IL-1ß, IL-6 and TNFα, corresponding to a 23% decrease in p38 phosphorylation. E-selectin and P-selectin expression was further reduced following DS-SILY20 treatment in both co-culture models. These results indicate that DS-SILY20 promotes EC health and that this decorin mimic could serve as a potential therapeutic to promote vessel healing following percutaneous coronary intervention (PCI). Copyright © 2015 John Wiley & Sons, Ltd.

In Vivo Functional Evaluation of Tissue-Engineered Vascular Grafts Fabricated Using Human Adipose-Derived Stem Cells from High Cardiovascular Risk Populations.

Many preclinical evaluations of autologous small-diameter tissue-engineered vascular grafts (TEVGs) utilize cells from healthy humans or animals. However, these models hold minimal relevance for clinical translation, as the main targeted demographic is patients at high cardiovascular risk such as individuals with diabetes mellitus or the elderly. Stem cells such as adipose-derived mesenchymal stem cells (AD-MSCs) represent a clinically ideal cell type for TEVGs, as these can be easily and plentifully harvested and offer regenerative potential. To understand whether AD-MSCs sourced from diabetic and elderly donors are as effective as those from young nondiabetics (i.e., healthy) in the context of TEVG therapy, we implanted TEVGs constructed with human AD-MSCs from each donor type as an aortic interposition graft in a rat model. The key failure mechanism observed was thrombosis, and this was most prevalent in grafts using cells from diabetic patients. The remainder of the TEVGs was able to generate robust vascular-like tissue consisting of smooth muscle cells, endothelial cells, collagen, and elastin. We further investigated a potential mechanism for the thrombotic failure of AD-MSCs from diabetic donors; we found that these cells have a diminished potential to promote fibrinolysis compared to those from healthy donors. Together, this study served as proof of concept for the development of a TEVG based on human AD-MSCs, illustrated the importance of testing cells from realistic patient populations, and highlighted one possible mechanistic explanation as to the observed thrombotic failure of our diabetic AD-MSC-based TEVGs.

Molecular imaging of experimental abdominal aortic aneurysms.

Current laboratory research in the field of abdominal aortic aneurysm (AAA) disease often utilizes small animal experimental models induced by genetic manipulation or chemical application. This has led to the use and development of multiple high-resolution molecular imaging modalities capable of tracking disease progression, quantifying the role of inflammation, and evaluating the effects of potential therapeutics. In vivo imaging reduces the number of research animals used, provides molecular and cellular information, and allows for longitudinal studies, a necessity when tracking vessel expansion in a single animal. This review outlines developments of both established and emerging molecular imaging techniques used to study AAA disease. Beyond the typical modalities used for anatomical imaging, which include ultrasound (US) and computed tomography (CT), previous molecular imaging efforts have used magnetic resonance (MR), near-infrared fluorescence (NIRF), bioluminescence, single-photon emission computed tomography (SPECT), and positron emission tomography (PET). Mouse and rat AAA models will hopefully provide insight into potential disease mechanisms, and the development of advanced molecular imaging techniques, if clinically useful, may have translational potential. These efforts could help improve the management of aneurysms and better evaluate the therapeutic potential of new treatments for human AAA disease.

Mechanism of proton relaxation for enzyme-manipulated, multicomponent gold-magnetic nanoparticle chains.

Longitudinal and transverse relaxation times of multicomponent nanoparticle (NP) chains are investigated for their potential use as multifunctional imaging agents in magnetic resonance imaging (MRI). Gold NPs (ca. 5 nm) are arranged linearly along double-stranded DNA, creating gold NP chains. After cutting gold NP chains with restriction enzymes (EcoRI or BamHI), multicomponent NP chains are formed through a ligation reaction with enzyme-cut, superparamagnetic NP chains. We evaluate the changes in relaxation times for different constructs of gold-iron oxide NP chains and gold-cobalt iron oxide NP chains using 300 MHz (1)H NMR. In addition, the mechanism of proton relaxation for multicomponent NP chains is examined. The results indicate that relaxation times are dependent on the one-dimensional structure and the amount of superparamagnetic NP chains present in the multicomponent constructs. Multicomponent NP chains arranged on double-stranded DNA provide a feasible method for fabrication of multifunctional imaging agents that improve relaxation times effectively for MRI applications.