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.
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.
Biomaterial-free three-dimensional (3D) bioprinting is a relatively new field within 3D bioprinting, where 3D tissues are created from the fusion of 3D multicellular spheroids, without requiring ...biomaterial. This is in contrast to traditional 3D bioprinting, which requires biomaterials to carry the cells to be bioprinted, such as a hydrogel or decellularized extracellular matrix. Here, we discuss principles of spheroid preparation for biomaterial-free 3D bioprinting of cardiac tissue. In addition, we discuss principles of using spheroids as building blocks in biomaterial-free 3D bioprinting, including spheroid dislodgement, spheroid transfer, and spheroid fusion. These principles are important considerations, to create the next generation of biomaterial-free spheroid-based 3D bioprinters.
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.
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.
As the incidence of cardiovascular disease continues to climb worldwide, there is a corresponding increase in demand for surgical interventions involving vascular grafts. The current gold standard ...for vascular grafts is autologous vessels, an option often excluded due to disease circumstances. As a result, many patients must resort to prosthetic options. While widely available, prosthetic grafts have been demonstrated to have inferior patency rates compared with autologous grafts due to inflammation and thrombosis. In an attempt to overcome these limitations, many different materials for constructing vascular grafts, from modified synthetic nondegradable polymers to biodegradable polymers, have been explored, many of which have entered the translational stage of research. This article reviews these materials in the context of large animal models, providing an outlook on the preclinical potential of novel biomaterials as well as the future direction of vascular graft research.
Recent advances in the understanding and use of pluripotent stem cells have produced major changes in approaches to the diagnosis and treatment of human disease. An obstacle to the use of human ...induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for regenerative medicine, disease modeling and drug discovery is their immature state relative to adult myocardium. We show the effects of a combination of biochemical factors, thyroid hormone, dexamethasone, and insulin-like growth factor-1 (TDI) on the maturation of hiPSC-CMs in 3D cardiac microtissues (CMTs) that recapitulate aspects of the native myocardium. Based on a comparison of the gene expression profiles and the structural, ultrastructural, and electrophysiological properties of hiPSC-CMs in monolayers and CMTs, and measurements of the mechanical and pharmacological properties of CMTs, we find that TDI treatment in a 3D tissue context yields a higher fidelity adult cardiac phenotype, including sarcoplasmic reticulum function and contractile properties consistent with promotion of the maturation of hiPSC derived cardiomyocytes.
•3D microtissues used to probe biochemical maturation of hiPSC-cardiomyocytes (CMs).•Thyroid hormone, dexamethasone, and IGF-1 (TDI) improve CM maturation in 3D.•Gene expression and ultrastructure show improved adult cardiac phenotype.•Enhanced electrophysiological properties, contraction and ionotropic response
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.
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.
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.
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.
A key obstacle in the creation of engineered cardiac tissues of clinically relevant sizes is limited diffusion of oxygen and nutrients. Thus, there is a need for organized vascularization within a ...three-dimensional (3D) tissue environment. Human induced pluripotent stem cell (hiPSC)-derived early vascular cells (EVCs) have shown to improve organization of vascular networks within hydrogels. We hypothesize that introduction of EVCs into 3D microtissue spheroids will lead to increased microvascular formation and improve spheroid formation.
HiPSC-derived cardiomyocytes (CMs) were cocultured with human adult ventricular cardiac fibroblasts (FB) and either human umbilical vein endothelial cells (HUVECs) or hiPSC-derived EVCs for 72 h to form mixed cell spheroids. Three different groups of cell ratios were tested: Group 1 (control) consisted of CM:FB:HUVEC 70:15:15, Group 2 consisted of CM:FB:EVC 70:15:15, and Group 3 consisted of CM:FB:EVC 40:15:45. Vascularization, cell distribution, and cardiac function were investigated.
Improved microvasculature was found in EVC spheroids with new morphologies of endothelial organization not found in Group 1 spheroids. CMs were found in a core-shell type distribution in Group 1 spheroids, but more uniformly distributed in EVC spheroids. Contraction rate increased into Group 2 spheroids compared to Group 1 spheroids.
The triculture of CM, FB, and EVC within a multicellular cardiac spheroid promotes microvascular formation and cardiac spheroid contraction.
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