In this research, a tissue engineering approach was investigated for the fabrication of homogeneous polymer/cells scaffolds suitable for applications in the clinic, in particular for the treatment of coronary disease and atherosclerosis. Sodium alginate chemically modified with an RGD-containing peptide promoting cell attachment was chosen as the natural polymer for the preparation of matrix/cells constructs. Several conditions were investigated to optimise the derivatisation of the biomaterial, and no major dissimilarities were identified among alginates with different guluronic/mannuronic acid compositions and viscosities during the derivatisation process. Successful incorporation of the GRGDY-pentapeptide onto the alginate was achieved. Amino acid analysis, which allowed a reliable quantification of the degree of peptide attached, demonstrated that the complete pentapeptidic sequence was present in the alginate after completion of the derivatisation procedure. However, a significant loss of the peptide's final tyrosine residue was consistently observed during derivatisation. Studies were also performed using multipotent fibroblast-like plastic-adherent cells recovered from frozen bone marrow samples of adult patients, here defined as human "mesenchymal stem cells" (hMSCs). Prior to the cell immobilisation in the alginate matrix, the optimal conditions for cell expansion in monolayer culture were identified. Cell samples were assessed according to their percent viability at each expansion passage, doubling time, cell growth rate and specific glucose consumption rate. Overall, the highest cell growth kinetics, highest cell viabilities and lowest doubling times were observed for the combined use of serum-/bFGF-supplemented low-glucose DMEM medium and non-coated culture vessels in a 21% dissolved oxygen tension atmosphere. hMSCs expanded for four subsequent passages under these monolayer conditions proved to retain their undifferentiated state and their ability to differentiate toward multiple phenotypes. Finally, alginate/cells beads were prepared and maintained in the optimal culture conditions identified in the monolayer studies. Constructs prepared with either hMSCs or human foreskin fibroblasts in either unmodified alginate or alginate coupled with the GRGDY-pentapeptide were analysed. After immobilisation, both cell types failed to adhere to the matrix and to acquire their characteristic elongated, spindle-shaped morphology. Lack of cell proliferation and.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:498587 |
| Date | January 2008 |
| Creators | Pittarello, Ledo |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1445238/ |
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