Damage to intervertebral discs (IVD) is a leading contributor to disability worldwide. Current disc replacements have been pursued using artificial devices made from polymers and metal. However, artificial discs are typically unable to comply with the physiological motions of the spine and can damage adjacent tissue. This project focused on the regeneration of IVDs using bioprinted scaffolds. Firstly, the bioink options of collagen, alginate methylcellulose (alg/MC) and alginate–gelatin were screened. The alg/MC hydrogel was selected as the most conducive to accurate, stable and cell friendly scaffold production. Alg/MC was further characterised for bioprinting with bovine primary chondrocytes (BPCs) and the effect of MC sterilisation on cell compatibility investigated. It was identified that the alg/MC supported a chondrogenic phenotype, with a collagen type I: collagen type II gene expression ratio of 0.45:99.55 on day 21 of cell culture, and that autoclaved MC had a significant (P ≤ 0.05) beneficial effect relative to super critical CO2, based on DNA content (194% increase, in ng/scaffold) and matrix production (61% increase in safranin O stain intensity). Hydrostatic pressure (HP) stimuli was utilised with the aim to enhance the cartilage extracellular matrix (ECM). As a part of this, a meta-analysis review of HP literature was performed to identify key experimental parameters. Factors found to influence proteoglycan production to the greatest degree were identified as pressure magnitude (≤5-10 MPa), length of study (≥ 2 weeks) and HP application style (static over dynamic). Guided by the outcome of the meta-analysis an HP protocol was employed on alg/MC scaffolds laden with BPC or human IVD cells. The daily application of HP was shown to significantly (P ≤ 0.05) upregulate proteoglycan gene expression in both the BPC (78.1 ± 16.1 %) and human nucleus pulposus (hNP) cells (81.9 ± 26.4 %) relative to controls on day 21 of culture, supported also by histological staining and dimethyl methylene blue assay. Furthermore, the increase in proteoglycan (and associated water content) following HP was demonstrated using quantitative MRI (qMRI). Molecular quantities of water content (inferred by T2) and proteoglycan (measured by 1H MR spectroscopy) increased in HP treated samples relative to controls and were comparable to measurements taken from IVDs in vivo using the same methods. The results of this investigation suggested the potential of bioprinted chondrocyte-laden alg/MC with HP stimulation for engineering disc cartilage.
|Date of Award||Jul 2019|
|Supervisor||Derek Covill (Supervisor), Peter Bush (Supervisor), Mark Best (Supervisor), Nicholas G. Dowell (Supervisor) & Mara Cercignani (Supervisor)|
The generation of cartilage extracellular matrices using bioprinting
Hodder, E. (Author). Jul 2019
Student thesis: Doctoral Thesis