Ravi Birla

Optimizing a spontaneously contracting heart tissue patch with rat neonatal cardiac cells on fibrin gel

Engineered cardiac tissues have been constructed with primary or stem cell‐derived cardiac cells on natural or synthetic scaffolds. They represent a tremendous potential for the treatment of injured areas through the addition of tensional support and delivery of sufficient cells. In this study, 1–6 million (M) neonatal cardiac cells were seeded on fibrin gels to fabricate cardiac tissue patches, and the effects of culture time and cell density on spontaneous contraction rates, twitch forces and paced response frequencies were measured. Electrocardiograms and signal volume index of connexin 43 were also analysed. Patches of 1–6 M cell densities exhibited maximal contraction rates in the range 305–410 beats/min (bpm) within the first 4 days after plating; low cell density (1–3 M) patches sustained rhythmic contraction longer than high cell density patches (4–6 M). Patches with 1–6 M cell densities generated contractile forces in the range 2.245–14.065 mN/mm3 on days 4–6. Upon patch formation, a paced response frequency of approximately 6 Hz was obtained, and decreased to approximately 3 Hz after 6 days of culture. High cell density patches contained a thicker real cardiac tissue layer, which generated higher R‐wave amplitudes; however, low‐density patches had a greater signal volume index of connexin 43. In addition, all patches manifested endothelial cell growth and robust nuclear division. The present study demonstrates that the proper time for in vivo implantation of this cardiac construct is just at patch formation, and patches with 3–4 M cell densities are the best candidates. Copyright

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 30u2009000-70u2009000 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.

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 1.731u2009mV. 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.

32-Channel System to Measure the Electrophysiological Properties of Bioengineered Cardiac Muscle

The purpose of this study was to develop, assess, and validate a custom 32-channel system to analyze the electrical properties of 3-D artificial heart muscle (3D-AHM). In this study, neonatal rat cardiac cells were cultured in a fibrin gel to drive the formation of 3D-AHM. Once the tissues were fully formed, the customized electrocardiogram (EKG) sensing system was used to obtain the different electrophysiological characteristics of the muscle constructs. Additionally, this system was used to evaluate the electrical properties of native rat hearts, for comparison to the fabricated tissues and native values found in the literature. Histological evaluation showed extensive cellularization and cardiac tissue formation. EKG data analysis yielded time delays between the signals ranging from 0 to 7 ms. Optical maps exhibited slight trends in impulse propagation throughout the fabricated tissue. Conduction velocities were calculated longitudinally at 277.81 cm/s, transversely at 300.79 cm/s, and diagonally at 285.68 cm/s for 3D-AHM. The QRS complex exhibited an R-wave amplitude of 438.42 ± 36.96 μV and an average duration of 317.5 ± 16.5 ms for the tissue constructs. The data collected in this study provide a clearer picture about the intrinsic properties of the 3D-AHM while proving our systems efficacy for EKG data procurement. To achieve a viable and permanent solution, the bioengineered heart muscle must physiologically resemble native heart tissue as well as mimic its electrical properties for proper contractile function. This study allows us to monitor such properties and assess the necessary changes that will improve construct development and function.

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs

The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-h stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. An increase in contractile force was observed after the strain protocol of 10% stretch at 1xa0Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue.

Establishing the Framework for Tissue Engineered Heart Pumps

AbstractnDevelopment of a natural alternative to cardiac assist devices (CADs) will pave the way to a heart failure therapy which overcomes the disadvantages of current mechanical devices. This work provides the framework for fabrication of a tissue engineered heart pump (TEHP). Artificial heart muscle (AHM) was first fabricated by culturing 4xa0million rat neonatal cardiac cells on the surface of a fibrin gel. To form a TEHP, AHM was wrapped around an acellular goat carotid artery (GCA) and a chitosan hollow cylinder (CHC) scaffold with either the cardiac cells directly contacting the construct periphery or separated by the fibrin gel. Histology revealed the presence of cardiac cell layer cohesion and adhesion to the fibrin gel scaffold, acellular GCA, and synthesized CHC. Expression of myocytes markers, connexin43 and α-actinin, was also noted. Biopotential measurements revealed the presence of ~2.5xa0Hz rhythmic propagation of action potential throughout the TEHP. Degradation of the fibrin gel scaffold of the AHM via endogenous proteases may be used as a means of delivering the cardiac cells to cylindrical scaffolds. Further development of the TEHP model by use of multi-stimulus bioreactors may lead to the application of bioengineered CADs.

The design and fabrication of a three-dimensional bioengineered open ventricle

Current treatments in hypoplastic left heart syndrome (HLHS) include multiple surgeries to refunctionalize the right ventricle and/or transplant. The development of a tissue-engineered left ventricle (LV) would provide a therapeutic option to overcome the inefficiencies and limitations associated with current treatment options. This study provides a foundation for the development and fabrication of the bioengineered open ventricle (BEOV) model. BEOV molds were developed to emulate the human LV geometry; molds were used to produce chitosan scaffolds. BEOV were fabricated by culturing 30 million rat neonatal cardiac cells on the chitosan scaffold. The model demonstrated 57% cell retention following 4days culture. The average biopotential output for the model was 1615 µV. Histological assessment displayed the presence of localized cell clusters, with intercellular and cell-scaffold interactions. The BEOV provides a novel foundation for the development of a 3D bioengineered LV for application in HLHS.

Biosensors in Tissue and Organ Fabrication

We begin this chapter by looking at the role of sensors during tissue and organ fabrication. We provide a working model for sensor technology to support different parts of the tissue fabrication pathway. We next look at biological sensors in nature, with IGF-1 signaling as an example and study the intracellular signaling events which take place in response to IGF-1 signaling. Next, we look at the design requirements for sensors to support the culture and fabrication of 3D artificial tissue. The next three sections are dedicated to three specific sensing mechanisms, which include acoustic sensors, magnetic sensors and optical sensors. For each of the three sensor mechanism, we provide an overview of the theory, along with some applications to illustrate the principles of operation. We then look at flexible sensors and how they have been used for various applications and the potential role of flexible sensors in tissue engineering. We conclude this chapter by providing a case study from the Artificial Heart Laboratory; the case study is focused on the development of novel sensors to record the EKG properties of 3D artificial heart muscle.

16-Channel Flexible System to Measure Electrophysiological Properties of Bioengineered Hearts

As tissue engineering continues to mature, it is necessary to develop new technologies that bring insight into current paradigms and guide improvements for future experiments. To this end, we have developed a system to characterize our bioartificial heart model and compare them to functional native structures. In the present study, the hearts of adult Sprague–Dawley were decellularized resulting in a natural three-dimensional cardiac scaffold. Neonatal rat primary cardiac cells were then cultured within a complex 3D fibrin gel, forming a 3-dimensional cardiac construct, which was sutured to the acellular scaffold and suspended in media for 24–48xa0h. The resulting bioartificial hearts (BAHs) were then affixed with 16 electrodes, in different configurations to evaluate not only the electrocardiographic characteristics of the cultured tissues, but to also test the system’s consistency. Histological evaluation showed cellularization and cardiac tissue formation. The BAHs and native hearts were then evaluated with our 16-channel flexible system to acquire the metrics associated with their respective electrophysiological properties. Time delays between the native signals were in the range of 0–95xa0ms. As well, color maps revealed a trend in impulse propagation throughout the native hearts. After evaluation of the normal rat QRS complex we found the average amplitude of the R-wave to be 5351.48xa0±xa044.92xa0μV and the average QRS duration was found to be 10.61xa0±xa00.18xa0ms. In contrast, BAHs exhibited more erratic and non-uniform activity that garnered no appreciable quantification. The data collected in this study proves our system’s efficacy for EKG data procurement.

Introduction to Organ Fabrication

This chapter is designed to serve as an introduction to the field of xorgan fabrication. We begin by presenting a discussion on the chronic shortage of donor organs and the potential impact of bioartificial organs. We then discuss the field of organ fabrication in relation to other investigational research areas like genetic and protein engineering, cell transplantation and tissue engineering. We next describe specific steps in the organ fabrication pathway, which include cell sourcing, biomaterial synthesis, decellularization technology and bioreactor culture and conditioning. With this background in place, we describe specific examples from the literature, including fabrication of artificial liver, artificial lung and artificial pancreas. While the field of organ fabrication is promising, there are many scientific and technological challenges associated with every stage of the organ fabrication pathway. In the next section, we discuss some of these scientific and technological challenges that need to be overcome in order to move the field of organ fabrication forward. While bioartificial organs have the potential to create a significant impact in the field of organ transplantation, there are many other applications for bioartificial organs. In the next section, we describe one such potential application—the utilization of bioartificial organs as models for basic research. We conclude this chapter by describing the technology development pathway for bioartificial organs, including model development and validation studies and Phase I, Phase II and Phase III clinical trials.