Enhanced Viability of Endothelial Colony Forming Cells in Fibrin Microbeads for Sensor Vascularization

Enhanced vascularization at sensor interfaces can improve long-term function. Fibrin, a natural polymer, has shown promise as a biomaterial for sensor coating due to its ability to sustain endothelial cell growth and promote local vascularization. However, the culture of cells, particularly endothelial cells (EC), within 3D scaffolds for more than a few days is challenging due to rapid loss of EC viability. In this manuscript, a robust method for developing fibrin microbead scaffolds for long-term culture of encapsulated ECs is described. Fibrin microbeads are formed using sodium alginate as a structural template. The size, swelling and structural properties of the microbeads were varied with needle gauge and composition and concentration of the pre-gel solution. Endothelial colony-forming cells (ECFCs) were suspended in the fibrin beads and cultured within a perfusion bioreactor system. The perfusion bioreactor enhanced ECFCs viability and genome stability in fibrin beads relative to static culture. Perfusion bioreactors enable 3D culture of ECs within fibrin beads for potential application as a sensor coating

Soluble CD59 Expressed from an Adenovirus In Vivo Is a Potent Inhibitor of Complement Deposition on Murine Liver Vascular Endothelium

Inappropriate activation of complement on the vascular endothelium of specific organs, or systemically, underlies the etiology of a number of diseases. These disorders include atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, atherosclerosis, age-related macular degeneration, diabetic retinopathy, and transplant rejection. Inhibition of the terminal step of complement activation, i.e. formation of the membrane attack complex, using CD59 has the advantage of retaining the upstream processes of the complement cascade necessary for fighting pathogens and retaining complement's crucial role in tissue homeostasis. Previous studies have shown the necessity of membrane targeting of soluble CD59 in order for it to prove an effective inhibitor of complement deposition both in vitro and in vivo. In this study we have generated an in vivo model of human complement activation on murine liver vascular endothelium. This model should prove useful for the development of anti-complement therapies for complement-induced pathologies of vascular endothelium. Using this model, we have demonstrated the viability of a non membrane-targeted soluble CD59 to significantly inhibit complement deposition on the endothelium of murine liver vasculature when expressed in vivo from an adenovirus. This result, unanticipated based on prior studies, suggests that the use of non membrane-targeted sCD59 as an anti-complement therapy be re-visited

ENGINEERING OF CLINICAL-SCALE, IN VITRO VASCULARIZED BONE TISSUE FOR IMPLANTATION

Tissue engineering has been a rapidly expanding field dedicated to regeneration of tissue. The field has focused on application through combinations of 3 key components: cells, signals, and scaffolds. One ambitious combination of all three is the desire to engineer functional tissues in vitro to meet the clinical-demand of organ replacement. While major advances have been made, a critical obstacle that has yet to be overcome is the need to grow large volumes of complex 3D tissue. In this proposal, this issue is addresed in two ways: the use of a perfusion bioreactor system to culture 3D scaffolds to enhance mass transport, and engineering of a vascular network withing the scaffold for rapid perfusion once implanted in vivo. This thesis aims to address both aspects for bone tissue engineering by engineering pre-vascularized, mineralizing scaffolds that can be scaled up to clinically-relevant volumes by using a tubular perfusion bioreactor system (TPS). To address this, 3 aims were addressed. First, 3D culture of endothelial colony forming cells (ECFCs), a clinically-relevant cell population, was demonstrated utilizing fibrin gels within the TPS. The TPS allowed for viable culture of ECFCs within fibrin bead scaffold up to 1 week without a reduction in cell amount or genomic quality of the cells. Second, a co-culture model of angiogenesis utilizing ECFCs and mesenchymal stem cells (MSCs) was demonstrated to reproducibly form pre-formed vessel networks within a mineralizing fibrin scaffold. Data shows that MSC suspension concentration and fibrinogen concentration modulate the angiogenic response. Mineralization is demonstrated without the use of osteogenic media utilizing shear stress within the TPS. Finally, functionality of the pre-formed vessels is demonstrated following implantation to a SCID mouse model. Engineered human vessels showed anastasmosis to the host vasculature, with evidence of interconnected host and human vessel networks as well as formation of hybrid vessels. Additionally, evidence of mineralization within the scaffolds is maintained in TPS-cultured samples. In demonstrating these aims, future work should focus on fortifying the scaffold material to enable addressing implantation and persistence of clinically-relevant tissue volumes. In conclusion, pre-vascularization within bioreactor-cultured scaffolds represents a promising solution for future tissue engineering application.Ph.D. in Biomedical Engineering, May 201

Intraperitoneal injection of an adenovirus expressing GFP (AdCAGGFP) shows significant transduction of murine liver at 7 days post-injection.

<p>A representative image of a transverse section of mouse liver showing GFP transduction is presented. DAPI stain of cell nuclei of the same liver section is also shown. GFP+DAPI overlay shows that GFP expression was observed almost exclusively along the peritoneal membrane, with a few cells within the liver expressing GFP.</p

Intraperitoneal injection of an adenovirus expressing human sCD59 results in reduced deposition of human MAC on endothelial cells of liver vasculature.

<p>(A) Representative micrographs showing human MAC deposition on sinusoidal endothelial cells, as well as the endothelial cells of blood vessels in livers of mice perfused with mPECAM-1 antibody and NHS 7 days post-injection with an adenovirus expressing either sCD59 (AdCAGsCD59) or GFP (AdCAGGFP). Low and high magnification images are shown for each. The intensity and area of MAC staining is reduced in the AdCAGsCD59-treated mice. (B) The average MAC staining intensity of liver vasculature in AdCAGsCD59-injected mice is reduced by 62.1% (**p<0.01) relative to that of AdCAGGFP-injected mice (n = 8).</p

AdCAGsCD59 protects against human MAC deposition in the larger (non-capillary) blood vessels of the liver.

<p>(A) Representative images indicating human MAC staining on the endothelial cell layer of a large blood vessel of murine liver perfused with mPECAM-1 antibody and NHS 7 days post-injection with AdCAGsCD59 or AdCAGGFP. Corresponding brightfield images are also shown (inset). (B) The average MAC staining intensity per endothelium area of large liver vessels of AdCAGsCD59-injected mice is reduced by 41.4% (***p<0.001) relative to that of AdCAGGFP-injected mice (n = 8).</p

Intracardial delivery of mPECAM-1 antibody permits binding to a variety of murine tissues.

<p><u>Liver</u>: In the liver, mPECAM-1 antibody binds endothelial cells along the sinusoids (arrowhead), as well as those of larger blood vessels (arrow). <u>Retina</u>: In the posterior eyecup, mPECAM-1 binds endothelia of the choriocapillaris (arrowhead) and retinal vasculature (arrow). <u>Choroid</u>: A flatmount of the choroid/RPE harvested from Balb/C mice injected intracardially with mPECAM-1 antibody shows more clearly binding of the antibody to the choroidal endothelium. <u>Aorta</u>: In the aorta, mPECAM-1 antibody is observed to bind the endothelial cell layer on the luminal surface (higher magnification of boxed region shown in inset). Intracardial delivery of a generic anti-mouse (GAM) antibody does not result in labeling of endothelial cells in any of the tissues. G, Ganglion Cell Layer; I/O, Inner/Outer Nuclear Layer; C, Choroid.</p

Alteration of fibrin hydrogel gelation and degradation kinetics through addition of azo dyes

Fibrin is a degradable biopolymer with an excellent clinical safety profile. Use of higher mechanical strength fibrin hydrogels is limited by the rapid rate of fibrin polymerization. We recently demonstrated the use of higher mechanical strength (fibrinogen concentrations >30 mg/ml) fibrin scaffolds for surgical implantation of cells. The rapid polymerization of fibrin at fibrinogen concentrations impaired our ability to scale production of these fibrin scaffolds. We serendipitously discovered that the azo dye Trypan blue (TB) slowed fibrin gelation kinetics allowing for more uniform mixing of fibrinogen and thrombin at high concentrations. A screen of closely related compounds identified similar activity for Evans blue (EB), an isomer of TB. Both TB and EB exhibited a concentration dependent increase in clot time, though EB had a larger effect. While gelation time was increased by TB or EB, overall polymerization time was unaffected. Scanning electron microscopy showed similar surface topography, but transmission electron microscopy showed a higher cross-linking density for gels formed with TB or EB versus controls. Based on these data we conclude that addition of TB or EB during thrombin mediated fibrin polymerization slows the initial gelation time permitting generation of larger more uniform fibrin hydrogels with high-mechanical strength. © 2021 Wiley Periodicals LLC.Mayo Foundation for Medical Education and Research12 month embargo; first published: 11 May 2021This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Alteration of fibrin hydrogel gelation and degradation kinetics through addition of azo dyes

Fibrin is a degradable biopolymer with an excellent clinical safety profile. Use of higher mechanical strength fibrin hydrogels is limited by the rapid rate of fibrin polymerization. We recently demonstrated the use of higher mechanical strength (fibrinogen concentrations >30 mg/ml) fibrin scaffolds for surgical implantation of cells. The rapid polymerization of fibrin at fibrinogen concentrations impaired our ability to scale production of these fibrin scaffolds. We serendipitously discovered that the azo dye Trypan blue (TB) slowed fibrin gelation kinetics allowing for more uniform mixing of fibrinogen and thrombin at high concentrations. A screen of closely related compounds identified similar activity for Evans blue (EB), an isomer of TB. Both TB and EB exhibited a concentration dependent increase in clot time, though EB had a larger effect. While gelation time was increased by TB or EB, overall polymerization time was unaffected. Scanning electron microscopy showed similar surface topography, but transmission electron microscopy showed a higher cross-linking density for gels formed with TB or EB versus controls. Based on these data we conclude that addition of TB or EB during thrombin mediated fibrin polymerization slows the initial gelation time permitting generation of larger more uniform fibrin hydrogels with high-mechanical strength. © 2021 Wiley Periodicals LLC.Mayo Foundation for Medical Education and Research12 month embargo; first published: 11 May 2021This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]