AbstractsEngineering

Production of a safer, osteogenic, tissue engineered bone allograft

by Christopher Andrew Smith




Institution: University of Manchester
Department:
Year: 2015
Keywords: Bone; Bone allograft; Tissue engineering; Mesenchymal stem cell; Osteogenic; Acellular; Aging
Record ID: 1406763
Full text PDF: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:258288


Abstract

The use of allograft bone is effective in the treatment of large bone loss following tumour removal or surgery. However, it is not osteogenic due to a lack of viable osteogenic cells and the remaining marrow material is potentially harmful to the recipient. Sterilisation techniques, such as gamma irradiation, are routinely used to improve the safety of these grafts; however this fails to remove the immunogenic material and may diminish the bones innate properties. Thus, wash techniques are being developed to remove the deleterious marrow, whilst retaining the native properties of the bone so that through tissue engineering, pre-osteogenic cells may be added to aid osseointegration. To this end, this study utilised a novel wash process (developed by the National Health Service Blood and Transplant Tissue services (NHSBT)) on whole human femoral heads, to assess the resulting material’s suitability as a biological scaffold for bone tissue engineering (BTE). Following the wash process, marrow removal efficiency was analysed by biochemical testing and histological assessment, and biocompatibility of fresh-frozen and washed human bone was assessed using extract cytotoxicity assays with BM-MSCs. The results showed a marrow removal efficiency of 99.5%, leaving a material with only 16.7 ng DNA/100mg of dry material, and which histologically displayed minimal cellular content demonstrating that this was an efficient wash process producing an acellular biological scaffold material (<50ng DNA/100mg bone). Extract cytotoxicity testing indicated the material was biocompatible. Uniaxial compression to failure was performed on 1cm3 cubes using bone samples from mirrored location of bilaterally halved femoral heads, with one half washed, whilst the other was fresh-frozen. A random orientated “clinical” model was also utilised, with samples processed as fresh-frozen, washed and irradiated for comparative assessment. There was no significant change in the mechanical strength of the washed material compared to fresh-frozen samples or between sterilisation types, suggesting the washed bone was mechanically comparable to existing bone allograft stock. BM-MSCs from both young (≤50 years) and old donors (≥70 years) were seeded on washed bone cubes from young and old donors, and cultured in standard or osteogenic media. Samples were analysed at 0, 14 and 28 day timepoints for cell viability, osteogenic gene expression, alkaline phosphatase activity and histological analysis. Results indicated significant fold increases in cell metabolism at day 14 and 28, in both medium types compared to day 0 (p≤0.001). QRT-PCR data showed increased expression of osteogenic markers RUNX2 (p≤0.001), osteopontin (p≤0.001) and osteocalcin (p≤0.001) in both standard and osteogenic media with significantly higher RUNX2 and osteocalcin in osteogenic medium samples at day 28. Expression of osteogenic genes was significantly higher in young donor cells seeded on the washed bone compared to old donor cells, as was expression in BM-MSCs cultured on old donor bone…