Human mesenchymal stem cells for tissue engineering bone
My thesis hypothesises that cells isolated from human bone marrow could be stimulated to differentiate into osteoblasts and that, these cells when cultured on a scaffold could be used in the tissue engineering of bone, as the constructs could potentially be implanted into patients' bone defects resulting in increased healing. The multipotency of the bone marrow-isolated cells was assessed by the use of histological stains to show that they could be stimulated to differentiate into osteoblasts, chondrocytes and adipocytes. The cells were further characterised by Stro- 1 and, as a result, were defined as mesenchymal stem cells (MSCs). More specifically, it was shown that when the cells were cultured with osteogenic stimulants, the production of osteoblastic proteins, such as alkaline phosphatase, osteopontin and osteocalcin increased (P<0.05), indicating that they were differentiating into osteoblasts. The cells were shown to retain their multipotent potential, and could be manipulated by surfaces and culture supplements in vitro. The ability of MSCs to differentiate in this way is fundamental for their use in tissue engineering of bone. This concept was investigated by growing marrow-isolated human MSCs on 3-dimensional porous hydroxyapatite (HA) scaffolds. In this environment, MSCs were shown to differentiate into osteoblasts, producing extracellular bone matrix proteins, even without the use of osteogenic stimulants. In order to simulate living bone conditions, where osteoblasts are perfused with tissue fluid, a novel bioreactor was developed for the culture of the MSC-HA constructs. Use of the bioreactor, to perfuse MSCs with oxygenated medium, promoted 3-dimensional growth of cells and stimulated differentiation into active osteoblasts resulting in increased production of extracellular matrix. Furthermore, the flow of medium through the scaffold encouraged the movement of cells and increased penetration into the HA (P<0.05). Cryopreservation was shown to be an effective method of storage, as it did not alter the cell function, measured by proliferation and the ability to differentiate into osteoblasts. Thus, it can be used as a practical method for storage of MSCs between harvest and implantation. These results indicate that, as marrow-isolated MSCs can be cultured successfully on a scaffold, they could be used for the tissue engineering of bone and implanted into patients to repair bone defects.