Bio-engineered collagen-fibrin scaffolds for enhancing urological tissue regeneration

Urological tissue engineering offers alternative solutions for congenital and acquired diseases of the urinary tract, which may require reconstructive surgery. It evolves to overcome complications, which might be encountered after surgery. Therefore, tissue-engineered scaffolds hold great promise for addressing these complications. Collagen type I is the most abundant structural extracellular matrix protein in the human body. Fibrin involves in the clotting of blood within healing wounds, and has inherited integrin and growth factor binding sites. Bioreactors transmit tissue specific environmental cues to the engineered scaffolds. Moreover, the addition of bioactive molecules in combination with controlled delivery enhances the generation of functional tissues. In the first part of the thesis, a new flow bioreactor system, mimicking physiological flow conditions in the human ureter, was developed and optimized. Human urinary tract smooth muscle (hSMC) cells were integrated within compressed tubular collagen scaffolds, prior to seeding human urinary tract urothelial cell (hUC) on the luminal surface of the scaffolds. The ureter flow mimicking conditions supported an evident cell proliferation and differentiation for both cell types. Superior extracellular matrix (ECM) protein deposition on collagen tubes was observed under dynamic conditions. Hence, our flow bioreactor design allowed producing ECM-like and large-scale collagen tubes to address ureter tissue repairs. In the second part of the thesis, a recombinant human insulin-like growth factor 1 (IGF-1) variant was produced and was used to functionalize a multilayered scaffold consisting of fibrin layer, sandwiched between two collagen gels. The IGF-1 variant was covalently conjugated to fibrin and had a matrix metalloproteinase-cleavage tag to obtain cell-mediated delivery of the protein. The purified IGF-1 variant showed similar bioactivity compared to commercially available IGF-1. Finally, in vivo evaluation of these multilayered collagen-fibrin scaffolds was performed in a nude rat model to investigate their regenerative capacity. Four weeks post implantation results showed that the IGF-1 variant induced a dose-dependent host smooth muscle cell invasion to the implant area. Thus, this bioactive collagen-fibrin scaffold may offer an advanced approach to accelerate bladder regeneration. In the third part of thesis, a collagen-fibrin bead gel system was developed and characterized to evaluate its potential use as an injectable bulking material. A microfluidics-based fabrication method was employed to obtain the fibrin beads. This method enabled us to obtain fibrin beads with precisely controlled sizes and as well as conjugate recombinant proteins without affecting its biological activity. In vitro, fibrin beads had a positive effect on hSMC proliferation, viability, and migration. To ensure its injectability, fibrin beads were then embedded into collagen gel. After further testing in an in vivo model, this bioactive collagen-fibrin bead gel system might have a have great potential in long-term functional urinary muscle tissue restoration. Overall, this work highlights that collagen-fibrin hybrid materials can be designed as multifunctional scaffolds in different forms and shapes.