Optimizing cell seeding and retention in a three-dimensional bioengineered cardiac ventricle: The two-stage cellularization model.

Current cell seeding techniques focus on passively directing cells to a scaffold surface with the addition of dynamic culture to encourage cell permeation. In 3D tissue engineered constructs, cell retention efficiency is dependent on the cell delivery method, and biomaterial properties. Passive cell delivery relies on cell migration to the scaffold surface; biomaterial surface properties and porosity determine cell infiltration capacity. As a result, cell retention efficiencies remain low. The development of an effective two-stage cell seeding technique, coupled with perfusion culture, provides the potential to improve cellularization efficiency, and retention. This study, uses a chitosan bioengineered open ventricle (BEOV) scaffold to produce a two-stage perfusion cultured ventricle (TPCV). TPCV were fabricated by direct injection of 10 million primary rat neonatal cardiac cells, followed by wrapping of the outer scaffold surface with a 3D fibrin gel artificial heart muscle patch; TPCV were perfusion cultured for 3 days. The average biopotential output was 1.731 mV. TPCV cell retention following culture was approximately 5%. Cardiac cells were deposited on the scaffold surface and formed intercellular connections. Histological assessment displayed localized cell clusters, with some dissemination, and validated the observed presence of intercellular and gap-junction interactions. The study demonstrates initial effectiveness of our two-stage cell delivery concept, based on function and biological metrics. Biotechnol. Bioeng. 2016;113: 2275-2285. © 2016 Wiley Periodicals, Inc.

Establishing the framework to support bioartificial heart fabrication using fibrin-based three-dimensional artificial heart muscle.

Only 3000 heart transplants are performed in the USA every year, leaving some 30 000-70 000 Americans without proper care. Current treatment modalities for heart failure have saved many lives yet still do not correct the underlying problems of congestive heart failure. Tissue engineering represents a potential field of study wherein a combination of cells, scaffolds, and/or bioreactors can be utilized to create constructs to mimic, replace, and/or repair defective tissue. The focus of this study was to generate a bioartificial heart (BAH) model using artificial heart muscle (AHM), composed of fibrin gel and neonatal rat cardiac myocytes, and a decellularized scaffold, formed by subjecting an adult rat heart to a series of decellularization solutions. By suturing the AHM around the outside of the decellularized heart and culturing while suspended in media, we were able to retain functional cardiac cells on the scaffold as evinced by visible contractility. Observed contractility rate was correlated with biopotential measurements to confirm essential functionality of cardiac constructs. Cross-sections of the BAH show successful decellularization of the scaffold and contiguous cell-rich AHM around the perimeter of the heart.

State of the art in Purkinje bioengineering.

This review article will cover the recent developments in the new evolving field of Purkinje bioengineering and the development of human Purkinje networks. Recent work has progressed to the point of a methodological and systematic process to bioengineer Purkinje networks. This involves the development of 3D models based on human anatomy, followed by the development of tunable biomaterials, and strategies to reprogram stem cells to Purkinje cells. Subsequently, the reprogrammed cells and the biomaterials are coupled to bioengineer Purkinje networks, which are then tested using a small animal injury model. In this article, we discuss this process as a whole and then each step separately. We then describe potential applications of bioengineered Purkinje networks and challenges in the field that need to be overcome to move this field forward. Although the field of Purkinje bioengineering is new and in a state of infancy, it holds tremendous potential, both for therapeutic applications and to develop tools that can be used for disease modeling.

Hybrid Interventions for Pulmonary Vein Stenosis: Leveraging Intraoperative Endovascular Adjuncts in Challenging Clinical Scenarios.

Background: Pediatric pulmonary vein stenosis (PVS) is often progressive and treatment-refractory, requiring multiple interventions. Hybrid pulmonary vein interventions (HPVIs), involving intraoperative balloon angioplasty or stent placement, leverage surgical access and customization to optimize patency while facilitating future transcatheter procedures. We review our experience with HPVI and explore potential applications of this collaborative approach. Methods: Retrospective chart review of all HPVI cases between 2009 to 2023. Results: Ten patients with primary (n = 5) or post-repair (n = 5) PVS underwent HPVI at median age of 12.7 months (range 6.6 months-9.5 years). Concurrent surgical PVS repair was performed in 7/10 cases. Hybrid pulmonary vein intervention was performed on 17 veins, 13 (76%) with prior surgical or transcatheter intervention(s). One patient underwent intraoperative balloon angioplasty of an existing stent. In total, 18 stents (9 bare metal [5-10 mm diameter], 9 drug eluting [3.5-5 mm diameter]) were placed in 16 veins. At first angiography (median 48 days [range 7 days-2.8 years] postoperatively), 8 of 16 (50%) HPVI-stented veins developed in-stent stenosis. Two patients died from progressive PVS early in the study, one prior to planned reintervention. Median time to first pulmonary vein reintervention was 86 days (10 days-2.8 years; 8/10 patients, 13/17 veins). At median survivor follow-up of 2.2 years (2.3 months-13.1 years), 1 of 11 surviving HPVI veins were completely occluded. Conclusions: Hybrid pulmonary vein intervention represents a viable adjunct to existing PVS therapies, with promising flexibility to address limitations of surgical and transcatheter modalities. Reintervention is anticipated, necessitating evaluation of long-term benefits and durability as utilization increases.

Recent advances in biological pumps as a building block for bioartificial hearts.

The field of biological pumps is a subset of cardiac tissue engineering and focused on the development of tubular grafts that are designed generate intraluminal pressure. In the simplest embodiment, biological pumps are tubular grafts with contractile cardiomyocytes on the external surface. The rationale for biological pumps is a transition from planar 3D cardiac patches to functional biological pumps, on the way to complete bioartificial hearts. Biological pumps also have applications as a standalone device, for example, to support the Fontan circulation in pediatric patients. In recent years, there has been a lot of progress in the field of biological pumps, with innovative fabrication technologies. Examples include the use of cell sheet engineering, self-organized heart muscle, bioprinting and in vivo bio chambers for vascularization. Several materials have been tested for biological pumps and included resected aortic segments from rodents, type I collagen, and fibrin hydrogel, to name a few. Multiple bioreactors have been tested to condition biological pumps and replicate the complex in vivo environment during controlled in vitro culture. The purpose of this article is to provide an overview of the field of the biological pumps, outlining progress in the field over the past several years. In particular, different fabrication methods, biomaterial platforms for tubular grafts and examples of bioreactors will be presented. In addition, we present an overview of some of the challenges that need to be overcome for the field of biological pumps to move forward.

Semi-Automated Construction of Patient-Specific Aortic Valves from Computed Tomography Images.

This paper presents a semi-automatic method for the construction of volumetric models of the aortic valve using computed tomography angiography images. Although the aortic valve typically cannot be segmented directly from a computed tomography angiography image, the method described herein uses manually selected samples of an aortic segmentation derived from this image to inform the construction. These samples capture certain physiologic landmarks and are used to construct a volumetric valve model. As a demonstration of the capabilities of this method, valve models for 25 pediatric patients are created. A selected valve anatomy is used to perform fluid-structure interaction simulations using the immersed finite element/difference method with physiologic driving and loading conditions. Simulation results demonstrate this method creates a functional valve that opens and closes normally and generates pressure and flow waveforms that are similar to those observed clinically.

Current state of the art in hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is a complex congenital heart condition in which a neonate is born with an underdeveloped left ventricle and associated structures. Without palliative interventions, HLHS is fatal. Treatment typically includes medical management at the time of birth to maintain patency of the ductus arteriosus, followed by three palliative procedures: most commonly the Norwood procedure, bidirectional cavopulmonary shunt, and Fontan procedures. With recent advances in surgical management of HLHS patients, high survival rates are now obtained at tertiary treatment centers, though adverse neurodevelopmental outcomes remain a clinical challenge. While surgical management remains the standard of care for HLHS patients, innovative treatment strategies continue to be developing. Important for the development of new strategies for HLHS patients is an understanding of the genetic basis of this condition. Another investigational strategy being developed for HLHS patients is the injection of stem cells within the myocardium of the right ventricle. Recent innovations in tissue engineering and regenerative medicine promise to provide important tools to both understand the underlying basis of HLHS as well as provide new therapeutic strategies. In this review article, we provide an overview of HLHS, starting with a historical description and progressing through a discussion of the genetics, surgical management, post-surgical outcomes, stem cell therapy, hemodynamics and tissue engineering approaches.

Fabrication of functional cardiac, skeletal, and smooth muscle pumps in vitro.

Cardiovascular disease is one of the leading causes of death in the United States, and new treatments need to be developed in order to provide novel therapies. Tissue engineering aims to develop biologic substitutes that restore tissue function. The purpose of the current study was to construct cell-based pumps, which can be viewed as biologic left ventricular assist devices. The pumps were fabricated by culturing cardiac, skeletal, and smooth muscle cells within a fibrin gel and then each 3-D tissue construct was wrapped around a decellularized rodent aorta. We described the methodology for pump fabrication along with functional performance metric, determined by the intra-luminal pressure. In addition, histologic evaluation showed a concentric organization of components, with the muscle cells positioned on the outermost surface, followed by the fibrin gel and the decellularized aorta formed the innermost layer. Though early in development, cell-based muscle pumps have tremendous potential to be used for basic and applied research, and with further development, can be used clinically as cell-based left ventricular assist devices.

The Immune and Inflammatory Basis of Acquired Pediatric Cardiac Disease.

Children with acquired heart disease face significant health challenges, including a lifetime of strict medical management, multiple cardiac surgeries, and a high mortality risk. Though the presentation of these conditions is diverse, a unifying factor is the role of immune and inflammatory responses in their development and/or progression. For example, infectious agents have been linked to pediatric cardiovascular disease, leading to a large health burden that disproportionately affects low-income areas. Other implicated mechanisms include antibody targeting of cardiac proteins, infection of cardiac cells, and inflammation-mediated damage to cardiac structures. These changes can alter blood flow patterns, change extracellular matrix composition, and induce cardiac remodeling. Therefore, understanding the relationship between the immune system and cardiovascular disease can inform targeted diagnostic and treatment approaches. In this review, we discuss the current understanding of pediatric immune-associated cardiac diseases, challenges in the field, and areas of research with potential for clinical benefit.

Congenital Heart Disease: An Immunological Perspective.

Congenital heart disease (CHD) poses a significant global health and economic burden-despite advances in treating CHD reducing the mortality risk, globally CHD accounts for approximately 300,000 deaths yearly. Children with CHD experience both acute and chronic cardiac complications, and though treatment options have improved, some remain extremely invasive. A challenge in addressing these morbidity and mortality risks is that little is known regarding the cause of many CHDs and current evidence suggests a multifactorial etiology. Some studies implicate an immune contribution to CHD development; however, the role of the immune system is not well-understood. Defining the role of the immune and inflammatory responses in CHD therefore holds promise in elucidating mechanisms underlying these disorders and improving upon current diagnostic and treatment options. In this review, we address the current knowledge coinciding CHDs with immune and inflammatory associations, emphasizing conditions where this understanding would provide clinical benefit, and challenges in studying these mechanisms.

A methodological nine-step process to bioengineer heart muscle tissue.

Research in the field of heart muscle tissue engineering is focused on the fabrication of heart muscle tissue which can be utilized to repair, replace and/or augment functionality of damaged and/or diseased tissue. In the simplest embodiment, bioengineering heart muscle tissue constructs involves culture of cardiomyocytes within natural or synthetic scaffolds. Functional integration of the cells with the scaffold and subsequent remodeling lead to the formation of 3D heart muscle tissue and physiological cues like mechanical stretch, electrical stimulation and perfusion are necessary to guide tissue maturation and development. Potential applications for bioengineered heart muscle include use as grafts to repair or replace damaged tissue, as models for basic research and as tools for high-throughput screening of pharmacological agents. In this article, we provide a methodological process to bioengineer functional 3D heart muscle tissue and discuss state of the art and potential challenges in each of the nine-step tissue fabrication process.

Current State of the Art in Ventricle Tissue Engineering.

The field of ventricle tissue engineering is focused on bioengineering highly functioning left ventricles that can be used as model systems for basic cardiology research and for cardiotoxicity testing. In this article, we review the current state of the art in the field of ventricle tissue engineering and discuss different strategies that have been used to bioengineer ventricles. Based on this body of literature, there are now common themes in the field that provide guidance for future directives, also presented in this article.

Tissue engineering solutions to replace contractile function during pediatric heart surgery.

Pediatric heart surgery remains challenging due to the small size of the pediatric heart, the severity of congenital abnormalities and the unique characteristics of each case. New tools and technologies are needed to tackle this enormous challenge. Tissue engineering strategies are focused on fabricating contractile heart muscle, ventricles, Fontan pumps and whole hearts, and a transplantable tissue equivalent has tremendous implications in pediatric heart surgery to provide functional cardiac tissue. This technology will prove to be a game-changer in the field of pediatric heart surgery and provide a novel toolkit for pediatric heart surgeons. This review will provide insight into the potential applications of tissue engineering technologies to replace lost contractile function in pediatric patients with heart abnormalities.

3D bioprinting and its potential impact on cardiac failure treatment: An industry perspective.

3D printing technologies are emerging as a disruptive innovation for the treatment of patients in cardiac failure. The ability to create custom devices, at the point of care, will affect both the diagnosis and treatment of cardiac diseases. The introduction of bioinks containing cells and biomaterials and the development of new computer assisted design and computer assisted manufacturing systems have ushered in a new technology known as 3D bioprinting. Small scale 3D bioprinting has successfully created cardiac tissue microphysiological systems. 3D bioprinting provides an opportunity to evaluate the assembly of specific parts of the heart and most notably heart valves. With the continuous development of instrumentation and bioinks and a complete understanding of cardiac tissue development, it is proposed that 3D bioprinting may permit the assembly of a heart described as a total biofabricated heart.

3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells.

PURPOSE: The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. METHODS: hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. RESULTS: hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. CONCLUSION: Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

A Highly Conductive 3D Cardiac Patch Fabricated Using Cardiac Myocytes Reprogrammed from Human Adipogenic Mesenchymal Stem Cells.

PURPOSE: The objective of this study was to bioengineer 3D patches from cardiac myocytes that have been reprogrammed from human adipogenic mesenchymal stem cells (hADMSCs). METHODS: Human adipogenic mesenchymal stem cells (hADMSCs) were reprogrammed to form cardiac myocytes using transcription factors ETS2 and MESP1. Reprogrammed cardiac myocytes were cultured in a fibrin gel to bioengineer 3D patch patches. The effect of initial plating density (1-25 million cells per patch), time (28-day culture period) and treatment with 1 µM isoproterenol and 1 µM epinephrine were evaluated. RESULTS: 3D patches were fabricated using cardiac myocytes that have been reprogrammed from hADMSCs. Based on optimization studies, it was determined that 10 million cells were needed to bioengineer a single patch, that measured 2 × 2 cm2. Furthermore, 3D patches fabricated 10 million cells were stable in culture for up to 28 days. Treatment of 3D patches with 1 µM isoproterenol and 1 µM epinephrine resulted in an increase in the electrical properties, as measured by electrical impulse amplitude and frequency. An increase in the expression of mTOR, KCNV1, GJA5, KCNJ16, CTNNT2, KCNV2, MYO3, FOXO1 and KCND2 was noted in response to treatment of 3D patches with isoproterenol and epinephrine. CONCLUSION: Based on the results of this study, there is evidence to support the successful fabrication of a highly functional 3D patches with measurable electrical activity using cardiac myocytes reprogrammed from hADMSCs. 3D patches fabricated using optimal conductions described in this study can be used to improve the functional properties of failing hearts. Predominantly, in case of the infarcted hearts with partial loss of electrical activity, the electrical properties of the 3D patches may restore the electrical activity of the heart.

The Role of an IL-10/Hyaluronan Axis in Dermal Wound Healing.

Scar formation is the typical endpoint of postnatal dermal wound healing, which affects more than 100 million individuals annually. Not only do scars cause a functional burden by reducing the biomechanical strength of skin at the site of injury, but they also significantly increase healthcare costs and impose psychosocial challenges. Though the mechanisms that dictate how dermal wounds heal are still not completely understood, they are regulated by extracellular matrix (ECM) remodeling, neovascularization, and inflammatory responses. The cytokine interleukin (IL)-10 has emerged as a key mediator of the pro- to anti-inflammatory transition that counters collagen deposition in scarring. In parallel, the high molecular weight (HMW) glycosaminoglycan hyaluronan (HA) is present in the ECM and acts in concert with IL-10 to block pro-inflammatory signals and attenuate fibrotic responses. Notably, high concentrations of both IL-10 and HMW HA are produced in early gestational fetal skin, which heals scarlessly. Since fibroblasts are responsible for collagen deposition, it is critical to determine how the concerted actions of IL-10 and HA drive their function to potentially control fibrogenesis. Beyond their independent actions, an auto-regulatory IL-10/HA axis may exist to modulate the magnitude of CD4+ effector T lymphocyte activation and enhance T regulatory cell function in order to reduce scarring. This review underscores the pathophysiological impact of the IL-10/HA axis as a multifaceted molecular mechanism to direct primary cell responders and regulators toward either regenerative dermal tissue repair or scarring.

ß-Adrenergic stimuli and rotating suspension culture enhance conversion of human adipogenic mesenchymal stem cells into highly conductive cardiac progenitors.

Clinical trials using human adipogenic mesenchymal stem cells (hAdMSCs) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiomyocytes (CMs) or vasculature. Thus, reprogramming hAdMSCs into myocyte progenitors may fare better in future investigations. To reprogramme hAdMSCs into electrically conductive cardiac progenitor cells, we pioneered a three-step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, ß-adrenergic and hypoxic signalling induced in three-dimensional (3D) cardiospheres. In Stage 1, ETS2 and MESP1 activated NNKX2.5, TBX5, MEF2C, dHAND, and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells. Next, in Stage 2, ß2AR activation repositioned cardiac progenitors into de novo immature conductive cardiac cells, along with the appearance of RYR2, CAV2.1, CAV3.1, NAV1.5, SERCA2, and CX45 gene transcripts and displayed action potentials. In Stage 3, electrical conduction that was fostered by 3D cardiospheres formed in a Synthecon®, Inc. rotating bioreactor induced the appearance of hypoxic genes: HIF-1α/ß, PCG 1α/ß, and NOS2, which coincided with the robust activation of adult contractile genes including MLC2v, TNNT2, and TNNI3, ion channel genes, and the appearance of hyperpolarization-activated and cyclic nucleotide-gated channels (HCN1-4). Conduction velocities doubled to ~200 mm/s after hypoxia and doubled yet again after dissociation of the 3D cell clusters to ~400 mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5 to 1 m/s. Epinephrine stimulation of stage 3 cardiac cells in patches resulted in an increase in amplitude of the electrical wave, indicative of conductive cardiac cells. Our efficient protocol that converted hAdMSCs into highly conductive cardiac progenitors demonstrated the potential utilization of stage 3 cells for tissue engineering applications for cardiac repair.

Changes in gene expression during the formation of bioengineered heart muscle.

A three-dimensional bioengineered heart muscle (BEHM) construct model had been previously developed, exhibiting contractile forces up to 800 microN. The interest of this study was to determine gene expression levels of biologic markers involved in calcium-handling between BEHM, cell monolayer, and neonatal heart. Cardiac cells were isolated from one litter of F344 rats and organized into groups (n = 5): 4-, 7-, 10-day BEHM and cell monolayer; BEHM was evaluated for cell viability and contractility. Groups were then analyzed for mRNA expression of calcium-handling proteins: myosin heavy chain (MHC) alpha and beta, Sarcoplasmic reticulum Ca++ ATPase (SERCA) 2, phospholamban (PBL), and ryanodine receptor. BEHM exhibited electrically stimulated active force (208 +/- 12 microN day 4, 361 +/- 22 microN day 7, and 344 +/- 29 microN day 10) and no decrease in cell number. Real-time polymerase chain reaction (PCR) showed an increase in gene expression of all calcium-handling proteins in BEHM at 7 and 10 days compared with monolayers, for example, comparing BEHM to monolayer (7 and 10 days, respectively), MHC-alpha: 2600-fold increase and a 100-fold increase; MHC-beta: 70-fold increase at 10 days; ryanodine receptor: 74-fold increase at 10 days; SERCA: 19-fold increase and sixfold increase; PBL: 158-fold increase and 24-fold increase. It was concluded that a three-dimensional environment is a better culturing condition of cardiac cells than a monolayer. Also, BEHM constructs demonstrated a high similarity to a native myocardium, and is, thus, a good starting foundation for engineered heart muscle.

Cardiac cells implanted into a cylindrical, vascularized chamber in vivo: pressure generation and morphology.

We have previously described a model to implant dissociated cells into a cylindrical, vascularized bed in vivo to promote the formation of functional cardiac muscle constructs. We now investigate the cellular organization and the ability of the constructs to generate intra-luminal pressure. Primary cardiac cells were isolated from hearts of 2-3 day old rats, suspended in fibrin gel and inserted into the lumen of silicone tubing. The silicone tubing was then implanted around the femoral vessels in the groin region of recipient animals. After 3 weeks, the constructs were harvested, placed in an in vitro bath and cannulated via the incorporated femoral artery with a pressure transducer for evaluation of intra-luminal pressure dynamics. Histological evaluation showed the presence of a concentric ring of cardiac cells surrounding the femoral vessels. There was also a significant amount of collagen present around cardiac cells. In addition, we observed a significant amount of neovascularization of the explanted constructs. Electron microscopy showed the presence of longitudinally aligned fibers with a large number of gap junctions. Upon electrical stimulation of a single pulse (7 V, 1.2 ms), the constructs generated an intra-luminal pressure of 1.19 +/- 0.45 mmHg (n = 6). In addition, we were able to electrically pace the constructs at frequencies of 0.5-5 Hz. A Starling behavior of the inverse relation between baseline pressure and twitch pressure was observed. Cardiac cells implanted for 3 weeks into the cylindrical vascularized bed formed a tissue construct that demonstrated many of the contractile properties and morphology expected of functioning cardiac tissues.