Scaffold-Free Tubular Tissues Created by a Bio-3D Printer Undergo Remodeling and Endothelialization when Implanted in Rat Aortae

<div><p>Background</p><p>Small caliber vascular prostheses are not clinically available because synthetic vascular prostheses lack endothelial cells which modulate platelet activation, leukocyte adhesion, thrombosis, and the regulation of vasomotor tone by the production of vasoactive substances. We developed a novel method to create scaffold-free tubular tissue from multicellular spheroids (MCS) using a “Bio-3D printer”-based system. This system enables the creation of pre-designed three-dimensional structures using a computer controlled robotics system. With this system, we created a tubular structure and studied its biological features.</p><p>Methods and Results</p><p>Using a “Bio-3D printer,” we made scaffold-free tubular tissues (inner diameter of 1.5 mm) from a total of 500 MCSs (2.5× 10⁴ cells per one MCS) composed of human umbilical vein endothelial cells (40%), human aortic smooth muscle cells (10%), and normal human dermal fibroblasts (50%). The tubular tissues were cultured in a perfusion system and implanted into the abdominal aortas of F344 nude rats. We assessed the flow by ultrasonography and performed histological examinations on the second (n = 5) and fifth (n = 5) day after implantation. All grafts were patent and remodeling of the tubular tissues (enlargement of the lumen area and thinning of the wall) was observed. A layer of endothelial cells was confirmed five days after implantation.</p><p>Conclusions</p><p>The scaffold-free tubular tissues made of MCS using a Bio-3D printer underwent remodeling and endothelialization. Further studies are warranted to elucidate the underlying mechanism of endothelialization and its function, as well as the long-term results.</p></div

The system of lamination of the MCS (Bio-3D printer) (A) skewers the MCSs into needle-array according to a three-dimensional structure pre-designed on a computer system (C).

<p>It is possible to design a 3D tube-shaped structure, such as that plotted in green (B), on the workstation computers.</p

The rat aortae were stained with HE and CD31, respectively (A).

<p>The number of endothelial cells in the five rat aortae was counted and compared with those in the tubular tissues (B).</p

A phase contrast microscopy shows the spheroid morphology of a MCS (A).

<p>Spindle to polygonal cells are mixed in the MSC (B). Masson’s trichrome staining reveals the extensive collagenous extracellular matrix (ECM) as blue (C). vWF, CD31 and CellTracker Red-positive vascular endothelial cells are distributed to all parts of the MCS (D-F). SMA and desmin-positive HASMC intermingle with the other cell types (G,H).</p

An intra-operative photograph of end-to-end anastomosis between the tubular structure and the abdominal aorta of the nude rat (A).

<p>Color Doppler (left) and pulse Doppler (right) flow imaging of percutaneous ultrasonography show patent vessels on the fifth day after implantation (B).</p

Remodeling of the blood vessel (Post-implantation).

<p>The graft of post-implantation is patent and remodeled (A). The wall area and lumen area pre-implantation are shown in blue, post-implantation in red (B). The lumen area is enlarged (P = 0.032) and the wall area is decreased (P = 0.008) after implantation. The total wall area and lumen area shows no significant difference (C).</p

The schematic illustration of the bioreactor system (A).

<p>The vascular graft generated by the Bio-3D printer is cannulated by an outer 22 gauge intravenous catheter (SURFLO: Termo, Tokyo, Japan) which has side holes, and is perfused by culture medium for 2 days before implantation. A scaffold-free vascular graft is generated from the MCSs(B).</p

Histological examination of the luminal side of the vascular graft.

<p>At pre-implantation, the vascular endothelial cells distribute to the entire area of the graft. Conversely, after implantation, vWF, CD31 and CellTracker Red-positive endothelial cells are seen at the inner lumen of the vessel. Furthermore, the vascular endothelial cells cover the inner surface of the vessel more continuously on the fifth day than on the second day.</p

Histological examination of the vascular graft in a short axis cross-section (Pre-implantation).

<p>Hematoxylin and eosin staining reveals the internal and external margins are smooth and the pinhole by the pinholder are completely closed (A). Masson’s trichrome staining reveals the extensive collagenous ECM as blue (B). vWF (C) and CD31-positive vascular endothelial cells (D) are distributed to all parts of the graft.</p

Additional file 1 of Clinical effects of a selective urate reabsorption inhibitor dotinurad in patients with hyperuricemia and treated hypertension: a multicenter, prospective, exploratory study (DIANA)

Additional file 1: Table S1. Eligibility criteria for the DIANA study