HIGH FIDELITY CARDIAC VENTRICLE FABRICATED FROM POROUS ELASTOMER PROMOTES VASCULOGENESIS

BackgroundIn 2021, approximately 20.1 million people passed away from cardiovascular disease, accounting to almost one third of overall death tolls worldwide.[1] Whilst engineered cardiac constructs hold a great promise for minimally invasive cardiac regeneration, current cardiac models are not yet able to fully model native myocardium to serve as a regenerative therapy.[2] Engineered left ventricles (LV) recreate cardiac cavity and are able to model fluid ejection parameters such as ejection fraction.[3]Micro-scaled surface topographies on these scaffolds would help fiber orientation and hence pumping capability of the model.[4,5] Here, we propose that by fabrication of a highly porous, elastomeric scaffold, we can establish microvasculature within the cardiac construct in vitro and induce de novo vascularogenesis in vivo.Methodology Macro-and Micro-scaled porosity provide physical cues to promote vascularization.[6] To achieve this, a sacrificial negative mold shaped like adult heart was printed and a solution of polycaprolactone (PCL) was injected into the mold.Following solvent removal by lyophilization, and overnight wash of the sacrificial mold, scaffolds with 85-99% void have been created.(Figure 1a) The resultant scaffold was imaged by scanning electron microscopy (SEM) and Keyence microscopy revealing its topographical features.Mechanical properties of the scaffold were measured by biaxial and uniaxial stretching and as well as compression testing.It was found that a preseeding of scaffold with dental pulp stem cells (DPSC) or mixture of DPSC and endothelial cells (ECs) will provide better cardiomyocyte survival and retention.By using GFP-tagged ECs, progress of vessel development and maintenance, eccentricity, length and number of vessel junctions is tracked over time.Functionality of the cardiac tissue have also been measured by image analysis and electrical stimulation to obtain excitation threshold (ET), maximum capture rate (MCR), and force frequency relationships, as well as calcium handling with/without electrical stimulation.These scaffolds have been implanting in rat omentum evaluate their neovascularization, vascular anastamosis and cell retention post explant.Both in vitro and in vivo samples shall be stained for general filament, F-actin, cardiomyocytes, Troponin T and fibroblasts, Vimentin, general macrophages, CD68, M2 macrophage marker, CD206. ResultsOur results indicate that by utilizing this novel technique, we can get scaffolds with upto 99% porosity and 50% 12% surface porosity with elongated fibers and pores (circularity of 0.750.05).(Figure 1b) Uniaxial stretching of the scaffold has shown that apparent elasticity of the scaffold is around 25 to 75 kPa and its mechanical anisotropy of approximately 2. Additionally, 2-step seeding, and fibrin encapsulation has shown to provide improved cell retention, survival and elongation.These scaffolds were seeded with EC/DPSC/CMs to achieve beating scaffolds with elongated and oriented microvasculature.(Figure 1c) The scaffolds were implanted with different cell mixtures such as EC/DPSC/CMs, DPSC/CM or EC/DPSCs and observed formation of micro-and macro-vasculature around the implanted construct after 14 days.(Figure 1d) Conclusion Our results provide a proof-of-concept methodology for creation of highly porous tissue engineering constructs with complex details.It has proven to support vascularogenesis in vitro and neovascularization in vivo, hence having the potential for regenerative applications.