Characterisation, optimization and transplantation of a tissue engineered cardiac muscle flap

© 2014 Dr. Richard TeePublications included in thesis:Tee, R., Lokmic, Z., Morrison, W. A., & Dilley, R. J. (2010). Strategies in cardiac tissue engineering. ANZ Journal of Surgery, 80(10), 683-693. DOI: 10.1111/j.1445-2197.2010.05435.xTee, R., Morrison, W. A., Dusting, G. J., Liu, G-S., Choi, Y. S., Hsiao, S. T-F., et al. (2012). Transplantation of engineered cardiac muscle flaps in syngeneic rats. Tissue Engineering: Part A, 18(19-20), 1992-1999. DOI: 10.1089/ten.tea.2012.0151The field of tissue engineering presents a new means of generating tissues for reconstruction. Engineering functional myocardium de novo can potentially address the current challenges faced in the field of heart transplantation and congenital cardiac abnormalities. However, heart tissue is metabolically demanding, and therefore highly susceptible to ischaemia. As the main strategies to engineer myocardium rely on assembling cardiomyocytes in vitro using scaffolds (e.g. polymer based or hydrogel based) or in a form of scaffoldless cell-sheets, its survival through implantation in vivo relies on neovascularisation from the recipient bed. This presents a major hurdle for cardiac tissue engineers, as these ‘cardiac grafts’ are unlikely to survive, especially, in the harsh environment of ischaemic tissue post- myocardial infarction. Publications quantifying the survival of cardiac tissues after implantation in vivo are scarce but conservative estimates suggest that less than one-tenth of such grafted cardiac tissue will survive. Using an in vivo vascularisation approach at our laboratory, by placing a microsurgically fabricated arteriovenous loop into a polycarbonate chamber (AV loop tissue engineering chamber), cardiomyocytes suspended in a hydrogel-based scaffold assembled in vivo into an ‘engineered vascularised cardiac muscle flap’ that is potentially transplantable. While this concept seemed an attractive solution to the problem facing cardiac tissue engineering, several questions have yet to be investigated: 1. Will the tissue engineered cardiac muscle flap suffer minimal tissue loss, after transplantation? 2. Following transplantation to the epicardium, will the engineered cardiac muscle tissue integrate with the myocardium? In this thesis, a series of experiments were performed to answer these questions. The information obtained in the studies can be summarised as follows: A. Seeding syngeneic neonatal rat cardiomyocytes into the AV loop tissue engineering chamber with MatrigelTM as a scaffold, it is possible to generate a contractile cardiac muscle flap in Sprague Dawley rats. Immunohistological examination showed no signs of acute rejection. This implies the AV loop approach may be suitable when autologous cell sources are available for implantation. B. In a dose-response experiment, seeding 6 million cardiomyocytes per chamber was found to produce a small variance and a significant mean volume. This has important implications for the outcome of histomorphometric analysis of the flap. It was also observed that the cardiac tissue volume generated seemed to demonstrate a ‘dose-response trend’, that was not seen in other existing cardiac tissue engineered approaches. C. When investigating two isoforms of an enzyme system, NADPH oxidase, in hope of boosting angiogenesis to generate robust cardiac tissues, it was found that the angiogenic environment in the tissue engineering chamber was too complex to be altered by simply targeting a single factor. D. Subjecting the tissue engineered cardiac muscle flap to ischaemia time and conditions similar to that seen in the heterotopic rat heart model, the cardiac muscle flap did not show any quantitative loss or morphological changes when transplanted to an ectopic site. E. The small size of the Sprague Dawley rats and its anatomical features did not allow the cardiac muscle flap to be transplanted in an autologous fashion to the heart. A novel model using two syngeneic adult rats, allowed transplantation of the cardiac muscle flap based on a long pedicle from one rat to the other’s heart. The method was feasible and reproducible. Histological examination of the transplanted flap showed connective tissue integration of the flap with the host’s heart, however, the flap’s cardiac tissue remained separated from the myocardium by some collagenous tissue. In summary, a syngeneic cardiac muscle flap was generated in the AV loop tissue engineering chamber and the concept of a transplantable vascularised cardiac muscle flap was demonstrated. The novel allogeneic transplant model may have application as a platform for testing functionality of various cardiomyogenic stem cell sources. While much is there to overcome, this is a step forward in translating this approach from the bench to bedside