Gelatin Promotes Cell Retention Within Decellularized Heart Extracellular Matrix Vasculature and Parenchyma.

INTRODUCTION: Recellularization of organ decellularized extracellular matrix (dECM) offers a potential solution for organ shortage in allograft transplantation. Cell retention rates have ranged from 10 to 54% in varying approaches for reseeding cells in whole organ dECM scaffolds. We aimed to improve recellularization by using soluble gelatin as a cell carrier to deliver endothelial cells to the coronary vasculature and cardiomyocytes to the parenchyma in a whole decellularized rat heart. METHODS: Rat aortic endothelial cells (RAECs) were perfused over decellularized porcine aorta in low (1%) and high (5%) concentrations of gelatin to assess attachment to a vascular dECM model. After establishing cell viability and proliferation in 1% gelatin, we used 1% gelatin as a carrier to deliver RAECs and neonatal rat cardiomyocytes (NRCMs) to decellularized adult rat hearts. Immediate cell retention in the matrix was quantified, and recellularized hearts were evaluated for visible contractions up to 35 days after recellularization. RESULTS: We demonstrated that gelatin increased RAEC attachment to decellularized porcine aorta; blocking integrin receptors reversed this effect. In the whole rat heart gelatin (1%) increased retention of both RAECs and NRCMs respectively, compared with the control group (no gelatin). Gelatin was associated with visible contractions of NRCMs within hearts (87% with gelatin vs. 13% control). CONCLUSIONS: Gelatin was an effective cell carrier for increasing cell retention and contraction in dECM. The gelatin-cell-ECM interactions likely mediated by integrin.

Whole Cardiac Tissue Bioscaffolds.

Bioscaffolds serve as structures for cells in building complex tissues and full organs including heart. Decellularizing cardiac tissue results in cell-free extracellular matrix (ECM) that can be used as a cardiac tissue bioscaffold. The field of whole-heart tissue engineering has been revolutionized since the 2008 publication of the first perfusion-decellularized whole heart, and since then, studies have shown how decellularized cardiac tissue retains its native architecture and biochemistry following recellularization. Chemical, enzymatic, and physical decellularization methods preserve the ECM to varying degrees with the widely accepted standard of less than 50 ng/mg of double-stranded DNA present in decellularized ECM. Following decellularization, replacement of cells occurs via recellularization: seeding cells into the decellularized ECM structure either via perfusion of cells into the vascular conduits, injection into parenchyma, or a combination of perfusion and injection. Endothelial cells are often perfused through existing vessel conduits to provide an endothelial lining of the vasculature, with cardiomyocytes and other parenchymal cells injected into the myocardium of decellularized ECM bioscaffolds. Uniform cell density and cell retention throughout the bioscaffold still needs to be addressed in larger animal models of the whole heart. Generating the necessary cell numbers and types remains a challenge. Still, recellularized cardiac tissue bioscaffolds offer therapeutic solutions to heart failure, heart valve replacement, and acute myocardial infarction. New technologies allow for decellularized ECM to be bioprinted into cardiac bioscaffolds or formed into a cardiac hydrogel patch. This chapter reviews the advances made in decellularization and recellularization of cardiac ECM bioscaffolds with a discussion of the potential clinical applications of ECM bioscaffolds.

Non-volume-loaded heart provides a more relevant heterotopic transplantation model.

BACKGROUND: We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-volume-loaded (NL) and volume-loaded (VL) models were tested for their physiologic and immunologic properties to assess their suitability for transplant studies. METHODS: Syngeneic heterotopic heart transplants were performed according to the techniques previously described by Ono (NL) and Yokoyama (VL). Grafts were followed over 90 days with sequential echocardiography. Ex-vivo Langendorff perfusion was used to gain functional data. Allogeneic heart transplants were done to determine whether chronic allograft vasculopathy (CAV) develops at a different pace in both transplant models. RESULTS: The ischemic time during surgery was significantly longer using the VL model (p<0.001). The LV diameter of NL hearts decreased over time while that of the VL model significantly increased (p=0.004 on POD 90). Mean LV developed pressure and (dP/dt)max were significantly higher with the NL model (61.1+/-8.5 mmHg and 4261.7+/-419.6 mmHg/s) than with VL hearts (19.9+/-16.5 mmHg; p=0.011 and 924.8+/-605.6 mmHg/s; p<0.001). The mean weight of NL hearts (0.45+/-0.03 g) was significantly less than that of VL hearts (1.21+/-0.16 g, p<0.001). Histology of syngeneic NL grafts showed healthy, but partly atrophic myocardium, whereas the LV myocardium of VL hearts showed dilation and scarring typical for chronic ischemic injury. Heart allografts similarly developed CAV with luminal narrowing of 37.2+/-16.6% (NL) and 34.4+/-21.4% (VL), respectively by POD 90 (p=0.807). CONCLUSIONS: Since the coronary arteries in the VL model get perfused with partly deoxygenated blood, the myocardium suffers from chronic ischemic injury. We recommend using the NL model in preclinical transplant studies.

Heterotopic and orthotopic tracheal transplantation in mice used as models to study the development of obliterative airway disease.

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7). To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations. For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model. In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7). This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

LAD-ligation: a murine model of myocardial infarction.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment. Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia. The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.