Alternative cell source for autologous tissue engineering of intervertebral disc - discogenic differentiation

The spine consists of a series of bones with intervertebral discs between them. These washers or spacers consist of a particular type of cartilage which, besides acting as shock absorbers in the spine, also give it flexibility and allow the spine to bend and twist. Unfortunately, degeneration of the disc is common and can lead to back pain. Current methods of treatment are not always successful and we need to find better ways to treat degenerate discs and back pain. Tissue engineering is a promising approach because it uses living cells and they may be able to replace degenerate tissue with normal disc material. However, there are very few cells in the disc and they are often not very healthy or active. Also the environment inside the disc is unusual in that the cells have little food or oxygen, often in an acidic environment and have to carry a lot of mechanical load. Previous attempts to repair the disc by tissue engineering do not appear to have been very successful, although this approach shows promise for other, related cartilages. The aim of this project is to use different types of cells from other areas of the body such as from skin, bone marrow or fat. By growing them in various conditions cells change the way they grow and change the types of molecules they produce. We will identify what culture conditions might change skin, bone or fat cells into disc cells, so enabling them to make material suitable for replacing degenerate disc tissue. The culture conditions that are expected to influence the behaviour of these cells are: oxygen concentration, osmotic pressure, pH, concentration of lactic acid (a product of anaerobic respiration), mechanical load, nutrients and growth factors. Growth factors are molecules which influence the ability of cells to make new tissue; those that are of special importance here are the ones that encourage cells to make molecules that disc cells normally do. The work involved in this project will be carried out in the Centre for Spinal Studies at the Robert Jones & Agnes Hunt Orthopaedic Hospital in Oswestry (which forms part of the Institute of Science & Technology for Medicine at Keele University) and Oxford University. Staff in Oswestry and Oxford have a great deal of experience in treating back pain patients with degenerate discs and also in growing cells in the laboratory. Joint with BB/D004780/1.

Tissue engineering is currently being used clinically to repair cartilaginous tissues and is being used, in at least one centre, for the intervertebral disc. However, if an autologous cell source is to be used (to avoid transmission of disease and immunological reactions) disc cells may not be appropriate for this application. Nucleus pulposus has a very low cell density (4000 cells/cubic mm compared with 14,000 cells/cubic mm in articular cartilage - which is still regarded as low) and thus is not an appropriate cell source for repair of damaged discs. To elicit repair, implanted cells have to survive under the conditions existing in the disc where the ionic strength of the extracellular matrix is high (450-550 mOsm as compared with the usual 300 mOsm), partial pressures of oxygen are (1-10 per cent), pH is acidic and the cells are routinely exposed to high hydrostatic pressures (up to 35 Atmospheres) and tensile strains as the disc is loaded. Cell culture studies are usually carried out under 21 per cent oxygen at 300mOsm and at atmospheric pressure are unlikely to predict how cells will behave in the disc. Thus, it is proposed to investigate 1) whether other cell types are appropriate for tissue engineering of disc material (ie bone marrow derived cells, dermal fibroblasts and adipocytes) and 2) identify the optimum environmental conditions for differentiating and conditioning these cells before implantation. Environmental conditions to be investigated are: 1) partial pressure of oxygen; 2) osmolality; 3) lactate levels and pH; 4) nutrient levels; 5) growth factors e.g. TGF beta, FGF, IGF and OP-1 (BMP 7) and 6) mechanical loads (hydrostatic pressure, compression and shear). Choice of optimal environmental conditions may vary with cell type. The following outcome measures will be used to determine optimal environmental conditions: (i) All cultures will be screened for production of GAG/collagen to eliminate conditions producing cells unsuitable for disc repair. (ii) The remaining cells will be screened using a set of specific discogenic markers. Cells expressing appropriate discogenic markers will be assayed for 1) cell metabolism (both anabolic & catabolic processes); 2) cell turnover; 3) expression of extracellular matrix proteins and glycoproteins; 4) effect of co-culture of cell types and 5) matrix organisation and integration within explant cultures. These methods will first be established in bovine systems before establishing if human cells of the same types can be induced to differentiate in a similar fashion. Joint with BB/D004780/1.