The development of an implantable bioengineered tracheal scaffold for respiratory tissue regeneration
Creators: name: Khalid, Tehreem
Publisher: name: Royal College of Surgeons in Ireland
Year: 2023
Subjects: classificationCode: 320606
subject: Regenerative medicine (incl. stem cells)

description: Currently there is an unmet clinical need in respiratory medicine for the treatment of large tracheal defects. Damage associated with the upper airway resulting in narrowing, weakening or discontinuity is often a life-threatening condition. To date, there has been very little success in establishing a successful tracheal replacement option, but knowledge from these unsuccessful attempts has been gained in three key areas that need to be addressed by a singular scaffold in order to successfully replace tracheal tissue. Often attempts will fail because of necrosis due to poor-vascularisation within a construct, scaffold collapse or dislodgement due to mechanical mismatch and infection due to a lack of a respiratory epithelial layer. Tissue engineering techniques have the potential to address these major concerns and provide an innovative biocompatible scaffold for tracheal tissue replacement. The overall goal of this PhD project was to optimise a bilayered collagen-hyaluronic acid matrix (CHyA-B), which was previously developed as an in vitro respiratory model and has shown to facilitate the growth of respiratory epithelial cells on its non-porous film layer and respiratory fibroblast cells within its porous collagen sub-layer. Specifically, this thesis sought to investigate the feasibility of incorporating a 3D-printed (3DP) backbone of polycaprolactone (PCL) within the CHyA-B matrix to enhance its mechanical properties closer to native tracheal tissue and then assess this composite scaffold’s potential to support vascularisation and epithelisation. This thesis initially investigated different 3DP tubular designs, with mechanical analysis of the design’s compressive strength, flexibility, and compliancy. Through a process of elimination, a final design was obtained which generated mechanical properties close to native tissue, producing a high degree of flexibility but also maintaining its mechanical strength. This 3DP PCL design was then successfully incorporated within the CHyA-B matrix to produce a reinforced composite CHyA-B scaffold which resulted in an advantageous increase in mean pore diameter of the collagen-sublayer, ideal for cell growth and vascularisation. The thesis next focused on validating the reinforced composite scaffold’s vascularisation potential using a co-culture model. The scaffold successfully supported the formation and infiltration of vessel-like structures in vitro, confirming the 3DP fibres allowed for the formation of pre-engineered vasculature with no impediment. Additional validation was confirmed through the use of a chick chorioallantoic membrane (CAM) assay, in which a greater percentage of vascularisation area was observed within the reinforced composite scaffold over its non- reinforced counterpart. Having developed the reinforced composite scaffold and confirmed its ability to support vascularisation in vitro and within the CAM assay, the final study in this thesis investigated the potential of the scaffold to support the growth and differentiation of respiratory epithelial cells in tandem with the vascularisation co-culture in a novel tri-culture. The reinforced composite scaffold was found to have enhanced the synergistic effect of the tri-culture model with augmented respiratory differentiation and vascularisation found to have occurred. In conclusion, this thesis successfully addressed three major concerns of current tracheal replacement efforts and in turn developed a novel bilayered reinforced composite scaffold and tri-culture model effectively recapitulating native tissue. This work has begun to pave the way to solve the critical components required for clinically successful, next-generation implants for tracheal regeneration.