Design and fabrication of heart muscle using scaffold-based tissue engineering

Cardiac tissue engineering strategies are based on the development of functional models of heart muscle in vitro . Our research is focused on evaluating the feasibility of different tissue engineering platforms to support the formation of heart muscle. Our previous work was focused on developing three-dimensional (3D) models of heart muscle using self-organization strategies and biodegradable hydrogels. To build on this work, our current study describes a third tissue engineering platform using polymer-based scaffolding technology to engineer functional heart muscle in vitro . Porous scaffolds were fabricated by solubilizing chitosan in dilute glacial acetic acid, transferring the solution to a mold, freezing the mold at −80°C followed by overnight lyophilization. The scaffolds were rehydrated in sodium hydroxide to neutralize the pH, sterilized in 70% ethanol and cellularized using primary cardiac myocytes. Several variables were studied: effect of polymer concentration and chitosan solution volume (i.e., scaffold thickness) on scaffold fabrication, effect of cell number and time in culture on active force generated by cardiomyocyte-seeded scaffolds and the effect of lysozyme on scaffold degradation. Histology (hematoxylin and eosin) and contractility (active, baseline and specific force, electrical pacing) were evaluated for the cellularized constructs under different conditions. We found that a polymer concentration in the range 1.0–2.5% (w/v) was most suitable for scaffold fabrication while a scaffold thickness of 200 Μm was optimal for cardiac cell functionality. Direct injection of the cells on the scaffold did not result in contractile constructs due to low cell retention. Fibrin gel was required to retain the cells within the constructs and resulted in the formation of contractile constructs. We found that lower cell seeding densities, in the range of 1–2 million cells, resulted in the formation of contractile heart muscle, termed s mart m aterial i ntegrated h eart m uscle (SMIHMs). Chitosan concentration of 1–2% (w/v) did not have a significant effect on the active twitch force of SMIHMs. We found that scaffold thickness was an important variable and only the thinnest scaffolds evaluated (200 Μm) generated any measurable active twitch force upon electrical stimulation. The maximum active force for SMIHMs was found to be 439.5 ΜN while the maximum baseline force was found to be 2850 ΜN, obtained after 11 days in culture. Histological evaluation showed a fairly uniform cell distribution throughout the thickness of the scaffold. We found that lysozyme concentration had a profound effect on scaffold degradation with complete scaffold degradation being achieved in 2 h using a lysozyme concentration of 1 mg/mL. Slower degradation times (in the order of weeks) were achieved by decreasing the lysozyme concentration to 0.01 mg/mL. In this study, we provide a detailed description for the formation of contractile 3D heart muscle utilizing scaffold-based methods. We demonstrate the effect of several variables on the formation and culture of SMIHMs. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58652/1/31642_ftp.pd

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79060/1/j.1525-1594.2010.01007.x.pd

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

Functional Evaluation of Isolated Zebrafish Hearts

Abstract Traditional working heart preparations, based on the original Langendorff setup, are widely used experimental models that have tremendously advanced the cardiovascular field. However, these systems can be deceivingly complex, requiring the maintenance of pH with CO2, the delivery of oxygenated perfusate, and the need for extensive laboratory equipment. We have examined the feasibility of using isolated zebrafish (Danio rerio) hearts as an experimental model system, in which experimental procedures can be performed in the absence of the traditional requirements and sophisticated setup equipment. Isolated zebrafish hearts exhibited spontaneous contractile activity, could be electrically paced, and were responsive to pharmacologic stimulation with isoproterenol for 1.5 h after in vivo removal. Isolated zebrafish hearts offer a time- and cost-effective alternative to traditional Langendorff/working heart preparation models, and could be used to investigate cardiac function and repair.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63209/1/zeb.2008.0550.pd

In Vivo Conditioning of Tissue-engineered Heart Muscle Improves Contractile Performance

The ability to engineer cardiac tissue in vitro is limited by the absence of a vasculature. In this study we describe an in vivo model which allows neovascularization of engineered cardiac tissue. Three-dimensional cardiac tissue, termed “cardioids,” was engineered in vitro from the spontaneous delamination of a confluent monolayer of cardiac cells. Cardioids were sutured onto a support framework and then implanted in a subcutaneous pocket in syngeneic recipient rats. Three weeks after implantation, cardioids were recovered for in vitro force testing and histological evaluation. Staining for hematoxylin and eosin demonstrated the presence of viable cells within explanted cardioids. Immunostaining with von Willebrand factor showed the presence of vascularization. Electron micrographs revealed the presence of large amounts of aligned contractile proteins and a high degree of intercellular connectivity. The peak active force increased from an average value of 57 µN for control cardioids to 447 µN for explanted cardioids. There was also a significant increase in the specific force. There was a significant decrease in the time to peak tension and half relaxation time. Explanted cardioids could be electrically paced at frequencies of 1–5 Hz. Explanted cardioids exhibited a sigmoidal response to calcium and positive chronotropy in response to epinephrine. As the field of cardiac tissue engineering progresses, it becomes desirable to engineer larger diameter tissue equivalents and to induce angiogenesis within tissue constructs. This study describes a relatively simple in vivo model, which promotes the neovascularization of tissue-engineered heart muscle and subsequent improvement in contractile performance.  Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74650/1/j.1525-1594.2005.00148.x.pd

Development of a Novel Bioreactor for the Mechanical Loading of Tissue-Engineered Heart Muscle

Objective: In this study, we describe a novel bioreactor system to deliver controlled stretch protocols to bioengineered heart muscle (BEHM) constructs. Our primary objective was to evaluate the effect of mechanical stretch on the contractile properties of three-dimensional cardiac constructs in vitro. Methods: BEHMs were formed by culturing primary neonatal cardiac myocytes in a fibrin gel using a method previously developed in our laboratory. A custom bioreactor system was designed using SolidWorks (Concord, MA) and structural components were manufactured using fusion deposition modeling. We utilized the bioreactor to evaluate the effect of 2-, 6-, and 24-hour stretch protocols on the stretch-induced changes in contractile function of BEHMs. Results: We were able to demonstrate compatibility of the bioreactor system with BEHMs and were able to stretch all the constructs with zero incidence of failure. We found that loading the constructs for 2, 6, and 24 hours during a 24-hour period using a stretch protocol of 1 Hz, 10% stretch did not result in any significant change in the active force, specific force, pacing characteristics, and morphological features. Conclusions: In this study, we demonstrate compatibility of a novel bioreactor system with BEHMs and the stability of the BEHMs in response to stretch protocols.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63267/1/ten.2006.0359.pd

Contractile three-dimensional bioengineered heart muscle for myocardial regeneration

Tissue engineered heart muscle may be able to provide a treatment modality for early stage congestive heart failure. In this study, we describe a new method to engineer functional 3-dimensional heart muscle utilizing a biodegradable fibrin gel. Primary cardiac myocytes were isolated from hearts of 2- to 3-day-old rats and processed in one of the two ways. For the first method (layering approach), the cells were plated directly on the surface of a fibrin gel-coated on polydimethylsiloxane (PDMS) surfaces. The cells were cultured in growth media and the contractile performance evaluated after formation of 3-dimensional tissue constructs. For the second method (embedding approach), the cells were suspended with thrombin and plated on 35 mm tissue culture surfaces coated with PDMS. Fibrinogen was then added to the surface. Within 7 days after initial cell plating, a 3-dimensional tissue construct of cells derived from primary heart tissue (termed bioengineered heart muscle, BEHM) resulted for both approaches. Histological evaluation showed the presence of uniformly distributed cardiac cells throughout the BEHM, both in longitudinal and cross sections. The stimulated active force of BEHMs formed using the layering approach was 835.5 ± 57.2 ΜN ( N = 6) and 145.3 ± 44.9 ΜN ( N = 6) using the embedding approach. The stimulated active force was dependent on the initial plating density. It was possible to maintain the contractile function of BEHM in culture for up to 2 months with daily medium changes. The BEHMs exhibited inotropy in response to external calcium and isoproterenol and could be electrically paced at frequencies of 1–7 Hz. We describe a novel method to engineer contractile 3-dimensional cardiac tissue construct with a fourfold increase specific force compared to our previous model. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2007Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55978/1/31090_ftp.pd

Postoperative outcomes in oesophagectomy with trainee involvement

BACKGROUND: The complexity of oesophageal surgery and the significant risk of morbidity necessitates that oesophagectomy is predominantly performed by a consultant surgeon, or a senior trainee under their supervision. The aim of this study was to determine the impact of trainee involvement in oesophagectomy on postoperative outcomes in an international multicentre setting. METHODS: Data from the multicentre Oesophago-Gastric Anastomosis Study Group (OGAA) cohort study were analysed, which comprised prospectively collected data from patients undergoing oesophagectomy for oesophageal cancer between April 2018 and December 2018. Procedures were grouped by the level of trainee involvement, and univariable and multivariable analyses were performed to compare patient outcomes across groups. RESULTS: Of 2232 oesophagectomies from 137 centres in 41 countries, trainees were involved in 29.1 per cent of them (n = 650), performing only the abdominal phase in 230, only the chest and/or neck phases in 130, and all phases in 315 procedures. For procedures with a chest anastomosis, those with trainee involvement had similar 90-day mortality, complication and reoperation rates to consultant-performed oesophagectomies (P = 0.451, P = 0.318, and P = 0.382, respectively), while anastomotic leak rates were significantly lower in the trainee groups (P = 0.030). Procedures with a neck anastomosis had equivalent complication, anastomotic leak, and reoperation rates (P = 0.150, P = 0.430, and P = 0.632, respectively) in trainee-involved versus consultant-performed oesophagectomies, with significantly lower 90-day mortality in the trainee groups (P = 0.005). CONCLUSION: Trainee involvement was not found to be associated with significantly inferior postoperative outcomes for selected patients undergoing oesophagectomy. The results support continued supervised trainee involvement in oesophageal cancer surgery

Implantation models for cardiac tissue engineering.

Congestive heart failure (CHF) is a major medical challenge in developed countries. There are currently 4.8 million patients in the US living with CHF and the current economic cost is estimated to be $23.2 Billion annually. Current therapeutic strategies to treat CHF are limited to surgical transplantation, pharmacological intervention and mechanical cardiac support. Although these treatment options have significantly improved the quality of patient care, there are certain limitations to each of these approaches. Cardiac tissue engineered in vitro may provide an alternative treatment modality for CHF by providing transplantable tissue which can have direct applicability in cases of local myocardial infarct. The first part of the research describes a method for engineering 3-dimensional contractile cardiac tissue, termed cardioids. The cardioids exhibit several characteristics of normal cardiac physiology. Cardioids generate a peak active force of up to 75 muN and can be paced at physiological relevant frequencies of 1--5 Hz without notable fatigue. In addition, cardioids exhibit positive inotropy in response to ionic calcium and positive chronotropy in response to epinephrine. In the second part of this research, an alternative method for engineering 3-dimensional contractile cardiac tissue is described. The most attractive feature of the second method is that capillaries are engineered within the cardiac tissue construct. A significant improvement in contractility is observed with a peak active force of 800 muN. In the third part of this research we describe two implantation models to promote angiogenesis in previously engineered cardioids. In the first model, cardioids are housed in a support silicone chamber, the femoral artery and femoral vein are routed through the chamber and the entire chamber implanted in synergic recipients. In the second model, cardioids are anchored onto custom made acrylic support frames and then secured within a subcutaneous pocket in synergic recipients. Both models promoted angiogenesis within the engineered cardiac tissue resulting in a significant improvement in the contractility of cardioids.Ph.D.Applied SciencesBiological SciencesBiomedical engineeringCellular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124021/2/3121895.pd