Abstract

Cell-based therapies have a relatively long tradition in modern medicine. Since the 70s surgeons tried to treat malignant and non-malignant disease with direct injection of bone marrow cells. Other cell-based therapies have been proposed after these initial achievements, but it was only in the late eighties that a new concept of therapy, based on cells, has been organically developed. In that years, R. Langer, J. and C. Vacanti proposed the combined use of cells and materials (i.e., scaffolds) to repair tissues and organs, so overcoming the several problems associated with the use of transplants. They coined the term “tissue engineering” as “an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ”. The practical application of these concepts started at the Howard Green & Associates with the researches on cultured sheets of autologous epidermis transplanted to patients suffering from different types of skin lesions1. Other remarkable examples followed this initial attempt. Autologous osteoblast cells, taken from the periosteum and seeded into coral scaffolds, have been used to reconstruct the traumatically lost thumb of a patient. Occluded pulmonary arteries, replaced with a polycaprolactone-polyglycolic acid copolymers scaffold, seeded with own patient peripheral blood vessels cells, gave positive results. Similarly, isolated vascular smooth muscles and endothelial cells were used to reconstruct arteries. Another example is the attempted substitution of surgical bladder augmentation in favour of tissue engineered bladders made by collagen in which urothelial and smooth muscle cells have been seeded. The therapeutic approaches on which tissue engineering has been initially based can be divided in two major techniques: i) the use of scaffold embedded with cells that adhered and proliferated in it and ii) direct seeding of isolated cells in the injured part to promote regeneration. In more recent time, however, an new approach has been developed by a Japanese research group coordinated by prof. Okano. This has been named by him “cell sheet engineering”. The technique is based on the possibility to harvest an undamaged sheet of cells that can be directly transplanted to the injured organ and promote its recovery. Cell sheet engineering possess some advantages over the other techniques as will be clear from the next chapter. Nevertheless, it needs to be improved and, in particular, further studies are necessary to better comprehend the mechanisms by which the cell layer is harvested. This process is based on the behaviour of a “smart polymer” called poly(N-isopropylacrylamide) (PNIPAM) that is capable to trigger cells adhesion simply varying temperature. At 37 °C, cells can adhere and proliferate on substrates grafted with this peculiar polymer, but, once temperature is decreased, it modifies its structure causing cell detachment. If the cells are confluent, then a cell sheet can be harvested and, consequently, used for tissue engineering applications. The focus of the present work has been the study and characterization of smart substrates employed for cell sheet engineering. A general overview on tissue engineering and “cell sheet engineering” applications are summarized in the background (Chapter 1). The state of the art on the different substrates employed and the behaviour of smart polymer are introduced. The general introduction is concluded with the basic concepts on the synthesis route adopted (Chapter 2). The experimental section is divided in two distinct parts: 1) the first part (Chapter 3) is focused on PNIPAM. A deeper description of the characteristics and the applications for this polymer are presented in a brief introduction. Then, the synthesis and general characterization of the polymer are discussed. The smart properties of tethered PNIPAM are tested by in vitro cell cultures and cell sheets, harvested from the obtained samples, characterized. The behaviour of the outermost region of the PNIPAM-coating are deeply investigated by means of Wilhelmy plate technique. A possible model for the evolution of the observed phenomena is given. In the end, an analysis related to the influence of PNIPAM thickness is presented. In particular, the correlation between the polymer chins length and the smart behaviour is investigated by cell culture test and dynamic contact angle. 2) The second part of the of the work (Chapter 4) is dedicated to a different approach to obtain a cell sheet. In the initial section of the chapter, a possible electroactive substrate is examined as an alternative to PNIPAM. The unexpected results, however, led to a different strategy that is presented. Despite limited to a specific cell line, this method allowed for a simple cell sheet harvesting that is described. A possible application is proposed and the characterization of the substrates used for this approach are exposed. Finally, the biological response and the cell sheets obtained by this method are studied.