B. Holmes, P. Koti, N. Muselimyan, H. Asfour, N. Sarvazyan
George Washington University,
Keywords: 3D printing, bioprinting, cardiovascular
Summary:3D printing has been gaining popularity as a method of creating heart valves. However, to the best of our knowledge, no one was yet able to 3D print venous valves, particularly using cells or biocompatible materials. Venous valves are much smaller and more delicate structures as compared to heart valves, therefore they are particularly challenging to produce. Our studies represent the first steps toward creating biocompatible and implantable venous valves using 3D printing and tissue engineering tools. Toward this long-term goal, we pursued the following specific tasks. TASK 1 included exploration of several different valve designs, including a single flap valve, a two-flap valve, a floating ball valve and a tri-leaflet valve. Main dimensions included 5 mm diameter valve and a 1-mm wall thickness. Valves were printed from both silicone and polyurethane material, and demonstrated movable flaps in the presence of fluid. TASK 2 was to test nanoporous thermoplastic polyurethane (TPU) as a candidate material for 3D printing of venous valves or as a scaffold material for cardiac constructs. The TPU material was shown to support human umbilical vein endothelial cells (HUVECs) which form dense microvascular networks after 24 and 48 hours. In addition, when HUVECs were co-cultured with pre-fibroblastic stem cells they formed highly interconnected networks on 3D printed TPU structures. TASK 3 was to compare the viability of primary cardiac fibroblasts and their survival in multiple passages before and after an extrusion based 3D printing process. Analysis of cell viability was conducted with microscopy, immunocytochemistry, and bioluminescence imaging with Luciferin and CytoscanTM LDH assays. A BioBot 3D bioprinter was used to print cell laden BioGel, a cell printing material that is photocured using visible blue light. Finally, for our TASK 4 we designed 3D circular structures to support formation of a ring of tissue-engineered cardiac muscle suitable for implantation around the outer circumference of a vein. When placed around valve-containing vessel segments such self-beating rings can be potentially used to aid venous return. Scaffolds were designed as 1-mm high rings, with a 4-mm outer diameter and a 3.5-mm inner diameter. The wall of the ring also possessed a roughly 300 um channel designed to trap cells and foster cardiac tissue growth, as well as to encourage adequate fluid perfusion. Primary cardiac myocytes were cultured and seeded on samples printed on the BioBot from silicone and nano-porous TPU and coated in fibronectin for 24 hours. Cardiac cells with and without fibrin were observed adhering to structures on the inner and outer walls of the rings and in the channels, forming coherent beating cell structures. Our findings bring creation of implantable venous valves one step closer to reality. Ability to replace and repair these vascular structures will be a major development for a broad spectrum of ailments associated with chronic venous disease.