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Title: In situ engineering of skeletal tissues using self-assembled biomimetic scaffolds
Author: Burke, Julie Leigh
ISNI:       0000 0004 2724 3141
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2011
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The overall aim of this thesis was to establish whether selected Leeds' β-structure self- assembling peptides were suitable candidates for use in skeletal tissue engineering. This was addressed by first carrying out simple cytotoxicity studies on selected SAPs using an osteosarcoma cell line in vitro, followed by assessing extent of bone regeneration / repair and safety in vivo in a rabbit calvarial defect model. In vitro investigations revealed that the SAPs (P11-4, P11-8, P111-12, P11-1S, P11-16 and Pl1- 20) tested were cytocompatible, though the positively charged SAP P11-16 performed less well than other SAPs tested in the contact cytoxicity assay, possibly due to the presence of chemical contaminant introduced during the peptide purification process. Cell proliferation was generally superior on the negatively charged peptides compared with the positively charged peptides and cells appeared to thrive at the lower concentrations (30 mg/mL) of negatively charged SAPs compared with their behaviour on the positively charged peptides at similar concentrations. In vivo, SAPs P11-4, P11-8 and P11-20 were applied to critical size defects in rabbit calvaria. Puralvlatrix' and Bio-Oss" were used as positive controls and no treatment was used as the negative control. Calvaria were harvested after 3 days, 10 days, 28 days and 84 days and subjected to micro-CT. The calvarial samples were then decalcified and evaluated histologically and immunohistochemically. Application of the negatively charged P11-4 to cranial defects resulted in the most bone regeneration over 3 months. The other negatively charged peptide (P11-20) and the positively charged peptide (P11-8) resulted in a similar amount of bone regeneration as the positive control hydrogel, Puralvtatrix, and the empty control defects. The bone regenerated was of normal architecture and exhibited the usual patterns for the mature bone marker, osteocalcin. However, the patterns of bone regeneration associated with the different treatment applications varied, with Pn-4 treated sites showing a distinctive spicule-type bony repair which may reflect its superior mineral nucleating ability in vitro. Immunohistochemistry showed the presence of assembled SAP fibres within the defect sites at 3 days post treatment, with some indication of intracellular- SAP present at day 10. No evidence of fibrillar SAP was present at day 28 for any of the peptides used. All safety parameters investigated in the in vivo model (presence of localised tissue reaction, general well being of the animal, IgG levels, detection of specific anti-peptide antibodies, histological evidence of necrosis/inflammation/foreign body reaction, presence of amyloid deposits) were all negative, suggesting SAPs had low immunogenicity and were safe to use. In conclusion, this is the first time that these self-assembling peptides have been investigated in vivo. The results suggest that they as good as the market leads in inducing bone repair, their biocompatibilitv and low immunogenicity strongly support their further development for use in human tissue engineering applications.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available