Vascularization of PEGylated fibrin hydrogels increases the proliferation of human iPSC-cardiomyocytes.

Studies have long sought to develop engineered heart tissue for the surgical correction of structural heart defects, as well as other applications and vascularization of this tissue has presented a challenge. Recent studies suggest that vascular cells and a vascular network may have regenerative effects on implanted cardiomyocytes (CM) and nearby heart tissue separate from perfusion of oxygen and nutrients. The goal of this study was to test whether vascular cells or a formed vascular network in a fibrin-based hydrogel would alter the proliferation of human iPSC-derived CM. First, vascular network formation in a slowly degrading PEGylated fibrin hydrogel was optimized by altering the cell ratio of human umbilical vein endothelial cells to human dermal fibroblasts, the inclusion of growth factors, and the total cell concentration. An endothelial to fibroblast ratio of 5:1 and a total cell concentration of 1.1 × 106 cells/mL without additional growth factors generated robust vascular networks while minimizing the number of cells required. Using this optimized system, human iPSC-derived CM were cultured on hydrogels without vascular cells, hydrogels with unorganized encapsulated vascular cells, or hydrogels with encapsulated vascular cells organized into networks for 7 days. CM proliferation and gene expression were assayed following 7 days of culture on the hydrogels. The presence of vascular cells in the hydrogel, whether unorganized or in vascular networks, significantly increased CM proliferation compared to an acellular hydrogel. Hydrogels with unorganized vascular cells resulted in lower CM maturity evidenced by decreased expression of cardiac troponin t (TNNT2), myosin light chain 7, and phospholamban compared to hydrogels without vascular cells and hydrogels with vascular networks. Altogether, this study details a robust method of forming rudimentary vascular networks in a fibrin-based hydrogel and shows that a hydrogel containing endothelial cells and fibroblasts can induce proliferation in adjacent CM, and these cells do not hinder CM gene expression when organized into a vascular network.

Sustained in vivo perfusion of a re-endothelialized tissue engineered kidney graft in a human-scale animal model.

Introduction: Despite progress in whole-organ decellularization and recellularization, maintaining long-term perfusion in vivo remains a hurdle to realizing clinical translation of bioengineered kidney grafts. The objectives for the present study were to define a threshold glucose consumption rate (GCR) that could be used to predict in vivo graft hemocompatibility and utilize this threshold to assess the in vivo performance of clinically relevant decellularized porcine kidney grafts recellularized with human umbilical vein endothelial cells (HUVECs). Materials and methods: Twenty-two porcine kidneys were decellularized and 19 were re-endothelialized using HUVECs. Functional revascularization of control decellularized (n = 3) and re-endothelialized porcine kidneys (n = 16) was tested using an ex vivo porcine blood flow model to define an appropriate metabolic glucose consumption rate (GCR) threshold above which would sustain patent blood flow. Re-endothelialized grafts (n = 9) were then transplanted into immunosuppressed pigs with perfusion measured using angiography post-implant and on days 3 and 7 with 3 native kidneys used as controls. Patent recellularized kidney grafts underwent histological analysis following explant. Results: The glucose consumption rate of recellularized kidney grafts reached a peak of 39.9 ± 9.7 mg/h at 21 ± 5 days, at which point the grafts were determined to have sufficient histological vascular coverage with endothelial cells. Based on these results, a minimum glucose consumption rate threshold of 20 mg/h was set. The revascularized kidneys had a mean perfusion percentage of 87.7% ± 10.3%, 80.9% ± 33.1%, and 68.5% ± 38.6% post-reperfusion on Days 0, 3 and 7, respectively. The 3 native kidneys had a mean post-perfusion percentage of 98.4% ± 1.6%. These results were not statistically significant. Conclusion: This study is the first to demonstrate that human-scale bioengineered porcine kidney grafts developed via perfusion decellularization and subsequent re-endothelialization using HUVEC can maintain patency with consistent blood flow for up to 7 days in vivo. These results lay the foundation for future research to produce human-scale recellularized kidney grafts for transplantation.

Increasing salinity of fibrinogen solvent generates stable fibrin hydrogels for cell delivery or tissue engineering.

Fibrin has been used clinically for wound coverings, surgical glues, and cell delivery because of its affordability, cytocompatibility, and ability to modulate angiogenesis and inflammation. However, its rapid degradation rate has limited its usefulness as a scaffold for 3D cell culture and tissue engineering. Previous studies have sought to slow the degradation rate of fibrin with the addition of proteolysis inhibitors or synthetic crosslinkers that require multiple functionalization or polymerization steps. These strategies are difficult to implement in vivo and introduce increased complexity, both of which hinder the use of fibrin in research and medicine. Previously, we demonstrated that additional crosslinking of fibrin gels using bifunctionalized poly(ethylene glycol)-n-hydroxysuccinimide (PEG-NHS) slows the degradation rate of fibrin. In this study, we aimed to further improve the longevity of these PEG-fibrin gels such that they could be used for tissue engineering in vitro or in situ without the need for proteolysis inhibitors. It is well documented that increasing the salinity of fibrin precursor solutions affects the resulting gel morphology. Here, we investigated whether this altered morphology influences the fibrin degradation rate. Increasing the final sodium chloride (NaCl) concentration from 145 mM (physiologic level) to 250 mM resulted in fine, transparent high-salt (HS) fibrin gels that degrade 2-3 times slower than coarse, opaque physiologic-salt (PS) fibrin gels both in vitro (when treated with proteases and when seeded with amniotic fluid stem cells) and in vivo (when injected subcutaneously into mice). Increased salt concentrations did not affect the viability of encapsulated cells, the ability of encapsulated endothelial cells to form rudimentary capillary networks, or the ability of the gels to maintain induced pluripotent stem cells. Finally, when implanted subcutaneously, PS gels degraded completely within one week while HS gels remained stable and maintained viability of seeded dermal fibroblasts. To our knowledge, this is the simplest method reported for the fabrication of fibrin gels with tunable degradation properties and will be useful for implementing fibrin gels in a wide range of research and clinical applications.

Assessment of electrospun cardiac patches made with sacrificial particles and polyurethane-polycaprolactone blends.

Congenital heart defects (CHDs) are the leading cause of death in live-born infants. Currently, patches used in the repair of CHDs are exclusively inert and non-degradable, which increases the risk of arrhythmia, follow-up surgeries, and sudden cardiac death. In this preliminary study, we sought to fabricate biodegradable scaffolds that can support cardiac regeneration in the repair of CHDs. We electrospun biodegradable scaffolds using various blends of polyurethane (PU) and polycaprolactone (PCL) with and without sacrificial poly(ethylene oxide) (PEO) particles and assessed the mechanical properties, cell infiltration levels, and inflammatory response in vitro (surface cell seeding) and in vivo (subcutaneous mouse implant). We hypothesized that a blend of the two polymers would preserve the low stiffness of PU as well as the high cell infiltration observed in PCL scaffolds. The inclusion of PU in the blends, even as low as 10%, decreased cell infiltration both in vitro and in vivo. The inclusion of sacrificial PEO increased pore sizes, reduced Young's moduli, and reduced the inflammatory response in all scaffold types. Collectively, we have concluded that a PCL patch electrospun with sacrificial PEO particles is the most promising scaffold for further assessment as in our heart defect model.

Engineering Myocardium for Heart Regeneration-Advancements, Considerations, and Future Directions.

Heart disease is the leading cause of death in the United States among both adults and infants. In adults, 5-year survival after a heart attack is <60%, and congenital heart defects are the top killer of liveborn infants. Problematically, the regenerative capacity of the heart is extremely limited, even in newborns. Furthermore, suitable donor hearts for transplant cannot meet the demand and require recipients to use immunosuppressants for life. Tissue engineered myocardium has the potential to replace dead or fibrotic heart tissue in adults and could also be used to permanently repair congenital heart defects in infants. In addition, engineering functional myocardium could facilitate the development of a whole bioartificial heart. Here, we review and compare in vitro and in situ myocardial tissue engineering strategies. In the context of this comparison, we consider three challenges that must be addressed in the engineering of myocardial tissue: recapitulation of myocardial architecture, vascularization of the tissue, and modulation of the immune system. In addition to reviewing and analyzing current progress, we recommend specific strategies for the generation of tissue engineered myocardial patches for heart regeneration and repair.