Cell Modification in 3D: a new Paradigm in the Creation of Living Cell-Biomaterial Composites

Our aims are to develop new, improved materials and methods which will allow stem cells (mesenchymal; MSCs) to be manipulated to form bone tissue. We will do this using tiny (<100 nm; 1/1000 diameter human hair) calcium phosphate (hydroxyapatite; HAP) particles as vectors to carry specific biological molecules to the cells. To maximize the delivery of these chemical and genetic signals, the whole cell surface of each individual cell will be covered with the vectors (3D coating). Specifically, this will allow us to produce and grow self supporting, living bone tissue, either inside the body at the site of damage, or outside in culture dishes ready for implant. More generally, the improved efficiency and cost effectiveness of this approach will also enhance studies in the generation of other tissue types from MSCs (e.g. nerve and muscle) and in modifying other types of stem cell.Why mesenchymal stem cells?Stem cells have huge potential as therapeutic agents. Embryonic stem cells (ESC) have the potential to form all the major types of cell in the body, and are relatively easy to grow in culture. However, there are ethical and compatibility concerns with there use. Adult stem cells (ASC) can be harvested from specific tissue types (blood, nerves, skin), but the populations need to be expanded to get sufficient material for therapeutic use. In this regard, bone marrow mesenchymal stem cells (MSC) offer great hope for tissue engineering as methods for isolation and rapid cultivation are well established. They are natural precursors to bone, cartilage, fat and fibrous connective tissue formation. Thus they are already intensively studied as components of systems for replacing damaged bone tissues (e.g. restorative surgery). In addition the same person can be donor and recipient, thus alleviating the problems associated with ESCs.Why small hydroxyapatite particles?Hydroxyapatite is the chemical form of calcium phosphate found in bone, so it is compatible with the cells. The crystals of HAP in bone are also of a similar size (<100 nm). In addition, because the crystals are so small as well as coating the cell, some will be transported inside the cell. Thus the particles can be used to deliver information to the cell surface and interior.Why chemical and genetic signals?The key to using stem cells to regenerate tissue is the ability to persuade them to form the required type. There are two ways to manipulate these cells towards bone formation / direct genetic modification of the internal cell nucleus, or the use of indirect external stimuli such as chemicals secreted by other cells or present in the local cell environment, and physical contact with other cells.Why 3D coating?Bone-like cell behaviour is induced indirectly by adding expensive chemicals to the culture medium or adsorbing them onto the substrates. Direct genetic modification requires DNA to be delivered to the cell nucleus, typically by attachment to small carrier particles. For example, standard methods for gene delivery using HAP involve mixing the precursor chemicals together, then allowing the crystals that form to randomly settle on the cells like a snowstorm. There are two main factors which contribute to the inefficiency of both these current approaches; (a) the cells are adhered to a substrate, so not all the cell surface is available, and (b) the cells themselves are not targeted, so the additives are used at higher concentration than necessary. Both these problems should be alleviated by our proposed method.