Department of Physics, Chemistry and Biology (IFM)

Biomaterials Enhanced Regenerative Medicine

The goal of regenerative medicine is to repair failing tissues or organs or to replace failed ones, with the use of stem cells. More recently, it has been recognized that stem cells alone cannot affect the repairs, especially when there are physical gaps (Kureshi et al., 2009) Hence, biomaterials-enhanced stem cell therapies have now been the norm. A range of biomaterials have been developed and tested. These range from passive to highly interactive systems for cell delivery and/or to serve as scaffolds to recreate the target tissue or organ and restore function (Place et al., 2009).

Indeed, the new focus in regenerative medicine is the development of multi-functional, bio interactive scaffold to control stem cell proliferation and differentiation. On the flip side, to be able to develop and test a range of new biomaterials, reliable, reproducible stem cell systems are needed to make comparisons amongst a range of materials formulations or to differentiate between various topological formats of a single material.

Effect of Biomaterials Topology on Cell Proliferation and Differentiation

There has now been a range of reports that the topology of biomaterials has a role in the modulation of cellular adhesion, proliferation and differentiation. A range of techniques have also been developed to introduce different topologies, ranging for nanofabrication techniques such as moulding, etching, laser sculpting and electrospinning.

In electrospinning, ultrathin fibres are extruded across high voltage atmospheres and collected on a metal collecting plate. By varying different parameters, the fibre size (diameter), tightness of the mesh, aligned or non-aligned, can be produced. (Frenot and Chronakis, 2003) (Salto et al., 2008) (Nisbet et al., 2009).

The extracellular matrix (ECM) in tissues contains a range of fibrillar materials, and hence electrospinning techniques can be used to develop synthetic mimics of ECM. Electrospun meshes have topologies that can be divided into random fibres with high porosity and rough topology facilitating cell adhesion  (Decuzzi and Ferrari, 2010) and enabling nutrient exchange from the microcellular environment (Yang et al., 2004), or highly aligned fibres with closely packed smooth topography and guiding properties direct the cells to extend neuronal axons along the fibres (Kureshi et al., 2010) (Place et al., 2009). Moreover, finely oriented fibres have very large surface area which might also increase the rate of cell attachement and proliferation (Gupta et al., 2009) . However, there are no robust studies that specifically compare hydrogels/membranes vs electrospun fibres in very specific function.

Nerve Regeneration and Neural Progenitor Cells

The nervous system plays a complex and vital role in most of the physiological processes that undergo in a human body. Any severe, irreversible damage or injury to central and peripheral nervous system can result in neurodegenerative disorders, paralytic syndromes. There is no established therapy for complete cure for these disorders yet (Ghasemi-Mobarakeh et al., 2011) and hence, the remaining hope is on cell-based reparative therapies. Stem cell therapies and more recently, biomaterials-enhanced stem cell therapies are being studied.

Neural cell lines are often used to study the characteristics and differentiated features of authentic neurons (Francel et al., 1987). The Neuroblastoma Derived Cell line (NDC) is a rat- mouse hybrid neural precursor cell line generated by the fusion of mouse N18TG2 neuroblastoma cells and neonatal rat dorsal root ganglion neurons (DRG). (Wood et al., 1990). The cell line has the characteristics of a neural progenitor cell and differentiates under induction into neurons (Francel et al., 1987) or glia (Hackett et al., 2010). Furthermore, differentiated NDCs exhibit synaptic plasticity which is essential for neurotransmission (Hackett et al., 2010).

Being an immortalized line, NDCs can (in theory) be expanded indefinitely in culture and hence provide the homogeneity needed in comparative studies of biomaterials of different types, or of different topologies. Hence, NDCs could serve as a potential nerve source on the synthetic substrates for in vitro nerve regeneration.

Cardiac Regeneration and Cardiac Progenitor Cells

Myocardial infarction is a severe cardiovascular disorder and foremost reason for heart failure. The dead tissue on the infarcted region loses its contractility. Hence transplantation of cardiomyocytes might resolve further degeneration of cardiac function (Goumans et al., 2008) . The generation of cardiomyocytes, a terminally differentiated cell from the primitive cardiac stem cell is regarded as a compensation for life-threatening heart diseases. The heart was considered as terminally differentiated organ without self-renewable cells. Recently, a resident population cardiomyocyte Progenitor cells (CMPC) were effectively isolated and efficient differentiation into cardiomyocytes was carried out to achieve beating cardiac cells (Smits et al., 2009) .

Cardiac muscle specific stem or progenitors were first identified in the heart by Orlic et al., 2001. Since then, the cardiac stem cells (CSCs) have been characterized as self-renewing, pluripotent and can differentiate in vitro into specialized cells types of heart. Hence, autologous regeneration of myocardium can be possible in heart failure patients (Bearzi et al., 2007) . The regeneration of myocytes with contractile activity is also a justifying evidence for the regenerating potential of heart (Messina et al., 2004) .

The cardiac stem cells isolated from an adult heart, usually express stem cell surface antigens, Sca-1 (Stem cell antigen-1) and c-kit/CD117 (Matsuura et al., 2004) . The c-kit and Sca-1 expressing CSCs to give rise to myocytes, smooth muscles and endothelial cells Moreover, CSCs are clonogenic in nature (Beltrami et al., 2003) (Wang et al., 2006) . In the new born murine hearts, Cardiac stem cells express a transcription factor, Islet-1 (Isl-1) and the factors like GATA-4 expresses during the process of cardiogenesis (Leri et al., 2005) .

The Sca-1 expressing CSCs were essential for CSC proliferation, survival and regulation of cardiovascular differentiation (Tateishi et al., 2007) . The cardiac stem cells which highly expresses Sca-1 surface marker are subjected to the differentiation protocol induced by 5'-azacytidine or oxytocin and TGF-β1 (Transforming growth factor-β1) stimulation to produce cardiomyocytes (Chamuleau et al., 2009) . The 5'-azacytidine treatment stimulates sarcomere formation and initiates cluster formation before expressing the contractile activity of the cardiomyocytes (Goumans et al., 2008) .

In addition to this, several studies suggests that Sca-1 positive cardiac stem cells have great potential for regenerative therapy for cardiac injuries (Matsuura et al., 2004) (Wang et al., 2006) . The cardiac stem cells are demonstrated in several studies to be used for myocardial regeneration.

The current study aims at characterizing the isolated cells from murine heart as cardiac stem cells and differentiating the CSCs into cardiomyocytes in vitro for cardiac regeneration therapy. This is one of the preliminary studies to obtain optimal regenerative strategy to repair myocardial infarction.