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Production of a safer, osteogenic, tissue engineered bone allograft

Smith, Christopher Andrew

[Thesis]. Manchester, UK: The University of Manchester; 2015.

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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 compared to young bone. This implies that the washed bone was able to induce osteogenic differentiation in BM-MSCs, that young donor cells were better able to differentiate than old, and that old donor bone was better able to induce osteogenic activity. Additionally, patient-matched BM-MSCs and ASCs, and BM-MSCs and BM-MNCs were seeded onto washed bone cubes and cultured for 28 days in standard or osteogenic media, with gene expression and metabolic activity assessed. The washed bone was able to induce osteogenic differentiation of ASCs. Moreover, BM-MNCs when cultured on washed bone also expressed osteogenic genes, indicative of osteogenic differentiation. These results indicate the efficacy of a novel wash process in producing a biological acellular scaffold suitable for bone tissue engineering. Interestingly, data also suggests that the age of the cell donor and bone donor may effect osteogenic differentiation of seeded cells which has significant implications clinically.

Bibliographic metadata

Type of resource:
Content type:
Administered thesis
Form of thesis:
Traditional
Type of submission:
Doctoral level ETD - final
Thesis title:
Production of a safer, osteogenic, tissue engineered bone allograft
Degree type:
Doctor of Philosophy
Degree programme:
PhD Medicine (Inflammation & Repair)
Publication date:
2015-02-03T13:13:30
Institution:
The University of Manchester
Location:
Manchester, UK
Total pages:
278
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 compared to young bone. This implies that the washed bone was able to induce osteogenic differentiation in BM-MSCs, that young donor cells were better able to differentiate than old, and that old donor bone was better able to induce osteogenic activity. Additionally, patient-matched BM-MSCs and ASCs, and BM-MSCs and BM-MNCs were seeded onto washed bone cubes and cultured for 28 days in standard or osteogenic media, with gene expression and metabolic activity assessed. The washed bone was able to induce osteogenic differentiation of ASCs. Moreover, BM-MNCs when cultured on washed bone also expressed osteogenic genes, indicative of osteogenic differentiation. These results indicate the efficacy of a novel wash process in producing a biological acellular scaffold suitable for bone tissue engineering. Interestingly, data also suggests that the age of the cell donor and bone donor may effect osteogenic differentiation of seeded cells which has significant implications clinically.
Keyword(s):
Thesis main supervisor(s):
Thesis co-supervisor(s):
Degree grantor:
Language:
en

Institutional metadata

University researcher(s):
Academic department(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:258288
Created by:
Smith, Christopher
Created:
3rd February, 2015, 13:13:30
Last modified by:
Smith, Christopher
Last modified:
17th November, 2017, 08:44:32

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