Preclinical study of patient-specific cell-free nanofiber tissue-engineered vascular grafts using 3-dimensional printing in a sheep model

Abstract

Background: Tissue-engineered vascular grafts (TEVGs) offer potential to overcome limitations of current approaches for reconstruction in congenital heart disease by providing biodegradable scaffolds on which autologous cells proliferate and provide physiologic functionality. However, current TEVGs do not address the diverse anatomic requirements of individual patients. This study explores the feasibility of creating patient-specific TEVGs by combining 3-dimensional (3D) printing and electrospinning technology.

Methods: An electrospinning mandrel was 3D-printed after computer-aided design based on preoperative imaging of the ovine thoracic inferior vena cava (IVC). TEVG scaffolds were then electrospun around the 3D-printed mandrel. Six patient-specific TEVGs were implanted as cell-free IVC interposition conduits in a sheep model and explanted after 6 months for histologic, biochemical, and biomechanical evaluation.

Results: All sheep survived without complications, and all grafts were patent without aneurysm formation or ectopic calcification. Serial angiography revealed significant decreases in TEVG pressure gradients between 3 and 6 months as the grafts remodeled. At explant, the nanofiber scaffold was nearly completely resorbed and the TEVG showed similar mechanical properties to that of native IVC. Histological analysis demonstrated an organized smooth muscle cell layer, extracellular matrix deposition, and endothelialization. No significant difference in elastin and collagen content between the TEVG and native IVC was identified. There was a significant positive correlation between wall thickness and CD68⁺ macrophage infiltration into the TEVG.

Conclusions: Creation of patient-specific nanofiber TEVGs by combining electrospinning and 3D printing is a feasible technology as future clinical option. Further preclinical studies involving more complex anatomical shapes are warranted.

Keywords: 3D printing; Fontan circulation; cell-free tissue engineering; congenital heart disease; electrospun nanofibers; patient-specific; preclinical study; sheep model; tissue-engineered vascular graft.

FIGURE 1

Flow chart depicting the proposed process for manufacturing patient-specific tissue-engineered vascular grafts. A, Segmenting 3D image of the vasculature. B, Creating a patient-specific graft design from the preoperative computed tomography image using the computer-aided design system. C, Finalized mandrel. D, 3D-printed mandrel from stainless steel. E, Patient-specific nanofiber graft.

FIGURE 2

Study design of cell-free patient-specific nanofiber tissue engineered vascular graft. A and B, The dimension and shape of the thoracic inferior vena cava (IVC) was measured from angiography before surgery in the sheep model. C, An electrospinning mandrel was modeled by computer-aided design and subsequently 3D-printed. D, The nanofiber scaffold was electrospun onto the 3D-printed mandrel. E and F, A patient-specific cell-free nanofiber tissue-engineered vascular graft (TEVG) was implanted as an IVC interposition conduit in the sheep model. G and H, Scanning electron microscope images of the scaffold (G, 500×; H, 4000×). I, Intraoperative picture of the implanted TEVG. 3D, 3-dimensional.

FIGURE 3

Mechanical properties. The burst pressure (A) and compliance (B) of the native inferior vena cava (IVC) and patient-specific nanofiber tissue-engineered vascular graft (TEVG) both before and at 6 months after implantation. There was no significant difference in burst pressure and compliance between the native IVC and patient-specific nanofiber TEVG at 6 months. *P<.05.

FIGURE 4

Angiographic and quantitative biochemical analyses. A, Angiography of the patient-specific nanofiber tissue-engineered vascular grafts (TEVGs) at 6 months. *Indicates right atrium; arrows indicate anastomosis site of graft. B, Angiographic diameter change over time indicating narrowing of proximal, middle, and distal regions of patient-specific nanofiber TEVGs compared with native inferior vena cava (IVC) (6M/3M diameter). C, There was a statistically significant difference in TEVG pressure gradient between the 3- and 6-month time points. **P = .0045. D and E, The elastin and collagen content of the TEVG was not significantly different from the native IVC.

FIGURE 5

Representative hematoxylin and eosin photomicrographs of native inferior vena cava (IVC) (A) and patient-specific nanofiber tissue-engineered vascular graft (TEVG) (E). Only 2.09% ± 0.69% of the nanofiber scaffold material remained at 6 months. The TEVG endothelial cells and smooth muscle cells were mature and well-organized, and the smooth muscle cell layer was thicker than the native IVC in area, distribution, and density. Representative photomicrographs are shown for von Willebrand factor (B, native IVC; F, TEVG), α-smooth muscle actin (C, native IVC; G, TEVG), and myosin heavy chain (D, native IVC; H, TEVG). Scale bars: 2 mm for A and E; 20 μm for B and F; 100 μm otherwise.

FIGURE 6

Collagen and elastin deposition in the tissue-engineered vascular graft (TEVG) (E–H) at 6 months is similar to that of native inferior vena cava (IVC) (A–D) without ectopic calcification. Representative photomicrographs for picrosirius red (A and E), Masson’s trichrome (B and F), Hart’s (C, G), and von Kossa (D and H) stainings. Scale bars: 100 μm. I, Collagen content was similar in both the TEVG and native IVC.

FIGURE 7

Macrophage analysis and histomorphometry. A, Histomorphometric comparison of the wall thickness between native inferior vena cava (IVC) and patient-specific nanofiber tissue-engineered vascular graft (TEVG) revealed a statistically significant difference. **P = .0091. B, In addition, there was a significant positive correlation between the wall thickness of patient-specific nanofiber TEVGs and CD68⁺ macrophages/high-power field (HPF) in the patient-specific nanofiber TEVGs. P = .0037. C, Macrophage infiltration into the TEVGs was assessed by manual quantification of CD68⁺ cells. Scale bar: 20 μm.

VIDEO 1

Preclinical study of patient-specific cell-free nanofiber tissue-engineered vascular grafts (TEVGs) using 3D printing in a sheep model. Despite advances in surgical management of congenital heart disease, significant morbidity and mortality arise due to surgical complexity. Patient-specific graft design before surgery has the potential to overcome these challenges and improve patients’ quality of life. This study demonstrates our integrated approach of 3D imaging, 3D printing, and tissue engineering technology to design and create patient-specific vascular grafts before surgery in a sheep model. There were no significant differences in burst pressure and compliance between grafts and native inferior vena cava (IVC). The grafts showed native-like neotissue formation at 6 months after surgery with their scaffolds almost fully degraded, with only 2% of the initial scaffolds remaining on average. Extracellular matrix, elastin, and collagen production and endothelialization were similar in grafts and native IVC, with no vascular calcification. The presence of multilayer smooth muscle cells and a decreasing pressure gradient at 6 months compared with 3 months suggested graft remodeling. There was a significant positive relationship between graft wall thickness and CD68⁺ macrophage infiltration into the scaffold. We have demonstrated the feasibility of creating patient-specific nanofiber TEVGs using combined technology of electrospinning and 3D printing. Video available at: http://www.jtcvsonline.org/article/S0022-5223(16)31466-0/addons.