Chin Siang Ong

Tissue-Engineered Small Diameter Arterial Vascular Grafts from Cell-Free Nanofiber PCL/Chitosan Scaffolds in a Sheep Model

Tissue engineered vascular grafts (TEVGs) have the potential to overcome the issues faced by existing small diameter prosthetic grafts by providing a biodegradable scaffold where the patient’s own cells can engraft and form functional neotissue. However, applying classical approaches to create arterial TEVGs using slow degrading materials with supraphysiological mechanical properties, typically results in limited host cell infiltration, poor remodeling, stenosis, and calcification. The purpose of this study is to evaluate the feasibility of novel small diameter arterial TEVGs created using fast degrading material. A 1.0mm and 5.0mm diameter TEVGs were fabricated with electrospun polycaprolactone (PCL) and chitosan (CS) blend nanofibers. The 1.0mm TEVGs were implanted in mice (n = 3) as an unseeded infrarenal abdominal aorta interposition conduit., The 5.0mm TEVGs were implanted in sheep (n = 6) as an unseeded carotid artery (CA) interposition conduit. Mice were followed with ultrasound and sacrificed at 6 months. All 1.0mm TEVGs remained patent without evidence of thrombosis or aneurysm formation. Based on small animal outcomes, sheep were followed with ultrasound and sacrificed at 6 months for histological and mechanical analysis. There was no aneurysm formation or calcification in the TEVGs. 4 out of 6 grafts (67%) were patent. After 6 months in vivo, 9.1 ± 5.4% remained of the original scaffold. Histological analysis of patent grafts demonstrated deposition of extracellular matrix constituents including elastin and collagen production, as well as endothelialization and organized contractile smooth muscle cells, similar to that of native CA. The mechanical properties of TEVGs were comparable to native CA. There was a significant positive correlation between TEVG wall thickness and CD68+ macrophage infiltration into the scaffold (R2 = 0.95, p = 0.001). The fast degradation of CS in our novel TEVG promoted excellent cellular infiltration and neotissue formation without calcification or aneurysm. Modulating host macrophage infiltration into the scaffold is a key to reducing excessive neotissue formation and stenosis.

Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

We have developed a novel method to deliver stem cells using 3D bioprinted cardiac patches, free of biomaterials. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), fibroblasts (FB) and endothelial cells (EC) were aggregated to create mixed cell spheroids. Cardiac patches were created from spheroids (CM:FB:EC = 70:15:15, 70:0:30, 45:40:15) using a 3D bioprinter. Cardiac patches were analyzed with light and video microscopy, immunohistochemistry, immunofluorescence, cell viability assays and optical electrical mapping. Cardiac tissue patches of all cell ratios beat spontaneously after 3D bioprinting. Patches exhibited ventricular-like action potential waveforms and uniform electrical conduction throughout the patch. Conduction velocities were higher and action potential durations were significantly longer in patches containing a lower percentage of FBs. Immunohistochemistry revealed staining for CM, FB and EC markers, with rudimentary CD31+ blood vessel formation. Immunofluorescence revealed the presence of Cx43, the main cardiac gap junction protein, localized to cell-cell borders. In vivo implantation suggests vascularization of 3D bioprinted cardiac patches with engraftment into native rat myocardium. This constitutes a significant step towards a new generation of stem cell-based treatment for heart failure.

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

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.

3D bioprinting using stem cells

Recent advances have allowed for three-dimensional (3D) printing technologies to be applied to biocompatible materials, cells and supporting components, creating a field of 3D bioprinting that holds great promise for artificial organ printing and regenerative medicine. At the same time, stem cells, such as human induced pluripotent stem cells, have driven a paradigm shift in tissue regeneration and the modeling of human disease, and represent an unlimited cell source for tissue regeneration and the study of human disease. The ability to reprogram patient-specific cells holds the promise of an enhanced understanding of disease mechanisms and phenotypic variability. 3D bioprinting has been successfully performed using multiple stem cell types of different lineages and potency. The type of 3D bioprinting employed ranged from microextrusion bioprinting, inkjet bioprinting, laser-assisted bioprinting, to newer technologies such as scaffold-free spheroid-based bioprinting. This review discusses the current advances, applications, limitations and future of 3D bioprinting using stem cells, by organ systems.

Creation of cardiac tissue exhibiting mechanical integration of spheroids using 3D bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

Tissue engineered vascular grafts: current state of the field

ABSTRACT Introduction: Conventional synthetic vascular grafts are limited by the inability to remodel, as well as issues of patency at smaller diameters. Tissue-engineered vascular grafts (TEVGs), constructed from biologically active cells and biodegradable scaffolds have the potential to overcome these limitations, and provide growth capacity and self-repair. Areas covered: This article outlines the TEVG design, biodegradable scaffolds, TEVG fabrication methods, cell seeding, drug delivery, strategies to reduce wait times, clinical trials, as well as a 5-year view with expert commentary. Expert commentary: TEVG technology has progressed significantly with advances in scaffold material and design, graft design, cell seeding and drug delivery. Strategies have been put in place to reduce wait times and improve ‘off-the-shelf’ capability of TEVGs. More recently, clinical trials have been conducted to investigate the clinical applications of TEVGs.

Virtual Surgery for Conduit Reconstruction of the Right Ventricular Outflow Tract.

Purpose: Virtual surgery involves the planning and simulation of surgical reconstruction using three-dimensional (3D) modeling based upon individual patient data, augmented by simulation of planned surgical alterations including implantation of devices or grafts. Here we describe a case in which virtual cardiac surgery aided us in determining the optimal conduit size to use for the reconstruction of the right ventricular outflow tract. Description: The patient is a young adolescent male with a history of tetralogy of Fallot with pulmonary atresia, requiring right ventricle-to-pulmonary artery (RV-PA) conduit replacement. Utilizing preoperative magnetic resonance imaging data, virtual surgery was undertaken to construct his heart in 3D and to simulate the implantation of three different sizes of RV-PA conduit (18, 20, and 22 mm). Evaluation: Virtual cardiac surgery allowed us to predict the ability to implant a conduit of a size that would likely remain adequate in the face of continued somatic growth and also allow for the possibility of transcatheter pulmonary valve implantation at some time in the future. Subsequently, the patient underwent uneventful conduit change surgery with implantation of a 22-mm Hancock valved conduit. As predicted, the intrathoracic space was sufficient to accommodate the relatively large conduit size without geometric distortion or sternal compression. Conclusion: Virtual cardiac surgery gives surgeons the ability to simulate the implantation of prostheses of different sizes in relation to the dimensions of a specific patient’s own heart and thoracic cavity in 3D prior to surgery. This can be very helpful in predicting optimal conduit size, determining appropriate timing of surgery, and patient education.

Virtual surgical planning, flow simulation, and 3-dimensional electrospinning of patient-specific grafts to optimize Fontan hemodynamics

Background: Despite advances in the Fontan procedure, there is an unmet clinical need for patient‐specific graft designs that are optimized for variations in patient anatomy. The objective of this study is to design and produce patient‐specific Fontan geometries, with the goal of improving hepatic flow distribution (HFD) and reducing power loss (Ploss), and manufacturing these designs by electrospinning. Methods: Cardiac magnetic resonance imaging data from patients who previously underwent a Fontan procedure (n = 2) was used to create 3‐dimensional models of their native Fontan geometry using standard image segmentation and geometry reconstruction software. For each patient, alternative designs were explored in silico, including tube‐shaped and bifurcated conduits, and their performance in terms of Ploss and HFD probed by computational fluid dynamic (CFD) simulations. The best‐performing options were then fabricated using electrospinning. Results: CFD simulations showed that the bifurcated conduit improved HFD between the left and right pulmonary arteries, whereas both types of conduits reduced Ploss. In vitro testing with a flow‐loop chamber supported the CFD results. The proposed designs were then successfully electrospun into tissue‐engineered vascular grafts. Conclusions: Our unique virtual cardiac surgery approach has the potential to improve the quality of surgery by manufacturing patient‐specific designs before surgery, that are also optimized with balanced HFD and minimal Ploss, based on refinement of commercially available options for image segmentation, computer‐aided design, and flow simulations.

In vivo therapeutic applications of cell spheroids

Spheroids are increasingly being employed to answer a wide range of clinical and biomedical inquiries ranging from pharmacology to disease pathophysiology, with the ultimate goal of using spheroids for tissue engineering and regeneration. When compared to traditional two-dimensional cell culture, spheroids have the advantage of better replicating the 3D extracellular microenvironment and its associated growth factors and signaling cascades. As knowledge about the preparation and maintenance of spheroids has improved, there has been a plethora of translational experiments investigating in vivo implantation of spheroids into various animal models studying tissue regeneration. We review methods for spheroid delivery and how they have been utilized in tissue engineering experiments. We break down efforts in this field by organ systems, discussing applications of spheroids to various animal models of disease processes and their potential clinical implications. These breakthroughs have been made possible by advancements in spheroid formation, in vivo delivery and assessment. There is unexplored potential and room for further research and development in spheroid-based tissue engineering approaches. Regenerative medicine and other clinical applications ensure this exciting area of research remains relevant for patient care.

The use of 3D printing in cardiac surgery

In this issue, Chen et al. present a case report where a patient with a right-sided aortic arch, Kommerell’s diverticulum (KD) and an aberrant left subclavian artery underwent surgical repair, and the surgeons printed a patient-specific three-dimensional (3D) model before surgery to serve as a surgical guide. Using the 3D printed model, they managed to select the size of the frozen elephant trunk for implantation (32 mm) and visualize the aorta and its branches pre-operatively, to decide on the section to resect, reducing operative time. The patient made an uneventful recovery and postoperative computed tomography (CT) scan of the aortic arch demonstrated graft shape, position and patency.