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Title: Development of 3D tissue-engineered larynx using nanocomposite POSS-PCU material and stem cells
Author: Klanrit, P.
ISNI:       0000 0004 7230 234X
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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Background: Loss of laryngeal function severely affects the quality of life. To date, there are no optimised implants for laryngeal replacement or reconstruction; hence, it is still considered an unmet clinical need. The aim of this research is to develop a 3D scaffold made from POSS-PCU materials. This scaffold could then be assessed the biocompatibility with human primary bronchial epithelial cells (HBEC) and bone-marrow mesenchymal stem cells (BM-MSC) to produce tissue-engineered artificial larynx. Methods: 3D laryngeal scaffolds were fabricated from polyhedral oligomeric silsesquioxane – poly(carbonate-urea)urethane (POSS-PCU). The scaffolds were designed to be microporous with sodium bicarbonate (NaHCO3) particle size of 25 - 40 μm at different concentrations. This procedure created an interconnected microporous network and can be coated on the outer surface of casted POSS-PCU to create the 3D laryngeal frameworks. The materials were fully characterised and optimised for biocompatibility and voice function. HBEC and BM-MSC were seeded onto scaffolds and observed the viability as well as the characteristics after differentiation on the material such as morphology, and specific protein expressions via staining. Results: Comparison studies of mechanical strength showed no significant difference between porcine epithelial tissue and microporous POSS-PCU (P > 0.05), while the mechanical strength of the non-porous POSS-PCU was higher than the microporous samples and native epithelial tissue and cartilage (P < 0.05). The microporous POSS-PCU also responded to the air flow and generated the oscillating sound wave with the energy and fundamental frequency similar to the human range. Wettability analysis demonstrated the significant reduction in contact angle (θ o ) of the material after ethanol rinse (P < 0.05) which was similar to the commercial tissue-culture treated plastics (74°±3.32, P > 0.05). The material also showed the significant protein adsorption and enhanced cell attachment and growth in all samples with ethanol rinse. Primary HBEC and BM-MSC had significant growth on 3D porous scaffolds over 14 days (P < 0.05). Furthermore, the scaffold supported the differentiation of primary HBECs using air-liquid interface culture to develop mature epithelial cells with pseudostratified layer and migration into the POSS-PCU materials. SEM and immunofluorescence staining revealed the specific epithelial characteristics which were keratin 5 in the basal layer, keratin 18 on the top layer, epithelial tight junction, ciliated cells and mucin5AC production. BM-MSC also presented the growth and the differentiation on the material. Chondrogenic lineage properties had been observed through the change in cell morphology and the increased production of cartilaginous-like ECM such as sulphatedglycosaminoglycan (sGAG) and collagen. Conclusion: It had been demonstrated that the 3D scaffold made from POSS-PCU promotes cell adhesion, proliferation and differentiation with mature epithelial features as well as chondrogenic properties. This scaffold holds great promise to develop a tissueengineer-based larynx for laryngotracheal replacements.
Supervisor: Cousins, B. G. ; Birchall, M. ; Seifalian, A. M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available