Tesi di laurea magistrale
Fabrication and characterisation of bioartificial sponges for auricular cartilage engineering
Corso di studi
relatore Prof.ssa Ahluwalia, Arti Devi
correlatore Prof.ssa Danti, Serena
correlatore Prof.ssa Danti, Serena
- cartilage engineering
- bioartificial scaffold
- chondrogenic differentiation
- Tissue engineering
Data inizio appello
Auricle reconstruction due to congenital, post-infective or post-traumatic defects represents one of the most challenging procedure in the field of aesthetic and reconstructive surgery due to the highly complex three-dimensional anatomy of the external ear. In the last two decades tissue engineering strategies are under investigation as potential alternatives to overcome the shortcomings related to the harvesting of autogenous rib cartilage grafts employed in current gold standard surgical reconstruction procedure. In the present study, Poly(vinyl alcohol)/gelatin (PVA/G) sponges at different weight ratios [PVA/G 90/10, 80/20, 70/30 (w/w)%] were produced via emulsion and freeze-drying and finally cross-linked by exposure to glutaraldehyde vapors. Since the scaffolds aim to act as a preliminary extracellular matrix, the addition of a polysaccharide (i.e. alginate) was considered in this study [PVA/G/Alg 80/10/10, 90/5/5 (w/w)%]. However, the PVA/G/Alg foam did not maintain a suitable shape after freeze-drying. Differently, PVA/G sponges gave rise to highly porous, water stable and hydrophilic scaffolds, which were selected for further characterisation. Morphological, physico-chemical and biomechanical characterisation of PVA/G sponges showed interesting properties potentially useful for cartilage regeneration, such as round-shaped interconnected pores, high swelling capacity due to PVA and G interaction and chemical cross-linking, and essentially elastic behaviour. The PVA/G sponge 70/30 (w/w)% was selected since it presented a higher gelatin content and no significant differences in morphological, physico-chemical and mechanical properties when compared to PVA/G 80/20 (w/w)%. Biological studies were performed using the in vitro cell culture method. Since chondrocytes are difficult to isolate and expand in large numbers, bone marrow derived human mesenchymal stromal cells (hMSCs) were used, to be differentiated into chondrocytes. Different culture conditions were tested to optimise hMSC chondrogenesis on these scaffolds: commercial versus hand-made differentiation medium, undifferentiated versus pre-differentiated hMSC seeding, and static culture versus low-intensity ultrasound (US) stimulation. Cell viability throughout culture time proved that PVA/G is a suitable scaffold for hMSC adhesion, growth and chondrogenic differentiation. Experimental results highlighted an intense glycosaminoglycan (GAG), glycoprotein and collagen synthesis after three weeks of differentiation in a commercially available differentiative medium. Immunohistochemistry for chondrogenic markers revealed an early differentiation stage, characterised by the expression of Sox-9, a chondrogenic transcription factor, and type I collagen fibers. An initial expression of aggrecan was observed, while type II collagen and elastin were not detected at protein level. The application of US on cell/scaffold constructs proved an enhanced chondrogenic differentiation in terms of extracellular matrix deposition and a 30% higher type II collagen gene expression than those observed in the non-US stimulated counterparts. This study demonstrated that 70/30 PVA/G sponge is a suitable candidate for auricle reconstruction. Low-frequency US stimulation could represent a valuable tool to further improve chondrogenic differentation.
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