A Highly Perfused Tissue-engineered Construct for Gingival Tissue Regeneration using Degradable/Polar/Hydrophobic/Ionic Polyurethanes and A Dynamic Co-culture System
Advisor:
Santerre, Paul
Department:
Biomedical Engineering
Issue Date:
Jun-2015
Abstract (summary):
Gingival tissue engineering represents a promising method for regenerating the gingival lamina propria. In this thesis, a degradable polyurethane (degradable/polar/hydrophobic/ionic polyurethane (D-PHI)) scaffold was shown to support the proliferation and collagen production by human gingival fibroblasts (HGFs). As the gingival lamina propria is highly vascular, a perfusion bioreactor was designed to facilitate medium perfusion through HGF-seeded scaffolds. In the perfused (dynamic) culture, HGFs showed enhanced proliferation, metabolic activity and collagen production when compared with static cultures. These data suggest that the metabolic, phenotypic and differentiation state of HGFs were altered in dynamic cultures. Hence, the influence of the D-PHI material and perfusion on myofibroblast differentiation was explored. This study demonstrated that D-PHI does not enable myofibroblast differentiation over the 28-day culture. Moreover, it was shown that the perfused D-PHI scaffold modulated the fibroblast phenotype through complex mechanisms involving transforming growth factor (TGF)-beta1, basic fibroblast growth factor (FGF-2), beta1-integrin, and focal adhesion kinase, all of which were upregulated at the early time point (1 day) of the dynamic culture. These data provided new insights into the signalling pathways that are associated with the regulation of myofibroblast differentiation in a perfused system with elastomeric polymers such as D-PHI. Furthermore, using the bioreactor system developed in this thesis, a dynamic co-culture system with human umbilical vein endothelial cells (HUVECs) and HGFs was established. With the perfusion of medium, the production of vascular endothelial growth factor, an angiogenic factor, was induced in the HGFs. This study also investigated different co-culturing conditions (e.g. cell density, type of culture medium, cell seeding ratio) that can optimize cell growth, HUVEC cluster formation, angiogenic factor production, inhibition of myofibroblast differentiation, and collagen expression. It was found that at 80,000 cells/scaffold (12.95 mg, 80% porosity), a greater proportion of HGFs in the co-culture (i.e. 1:2 (HUVEC:HGF)) cultured in a 50/50 mixture of the HUVEC medium and HGF medium (by volume) can potentially induce HUVEC cluster formation, augment cell proliferation, and increase angiogenic factor production. The knowledge from this thesis could enable rational developments of the co-culture systems for building a functional, highly vascularized tissue-engineered construct for gingival tissue regeneration.
Permanent Link:
https://hdl.handle.net/1807/69300
Content Type:
Thesis
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