Development of a Biomaterials-based Functional Epithelial Graft for Tracheal Regeneration

Varma, Ratna 1, 2; Soon, Kayla 2; Karoubi, Golnaz 2; Waddell, Thomas 1, 2

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Latner Thoracic Surgery Research Laboratories, Toronto General Hospital

Tracheal injury, stenosis, and malignancy demand surgical management or tracheal reconstruction, however, the latter is an unmet clinical need as tracheal transplants fail due to the lack of a functioning epithelium and immune rejection. Tracheal immune rejection is largely associated with the epithelium, therefore we believe that the best method to optimize the use of donor tracheae is to decellularize only the epithelium and maintain the remaining tracheal structure. We aim to develop a donor-recipient hybrid tracheal model by de-epithelializing the donor trachea and engrafting a pre-developed biomaterials-based recipient epithelial graft. The absence of a functioning, ciliated, pseudostratified epithelium is a primary concern in tracheal regeneration. Clinically applied biomaterials demonstrate limited success in allowing proper airway epithelium growth and function. Promising biomaterials have not been studied systematically. Therefore, we present the first comparative study to determine the optimal biomaterial for this context.

Hyaluronan-Poly-ethylene glycol (HA-PEG), Gelatin, Chitosan-Collagen (CC), Collagen Vitrigel Membrane (CVM), and Silk Fibroin (SF) were screened to identify substrates that promote airway epithelium (cell line: BEAS-2B, and primary human tracheal epithelial cells: HTECs) attachment and function while possessing mechanical strength for transferability. Bulk degradation was assessed in the presence of all combinations of collagenase, protease XIV, and lysozyme, and initial Young’s moduli were measured. Cell attachment (vinculin), tight junction (zonula occludins-1) formation and differentiation of HTECs into ciliated (acetylated a-tubulin) in air-liquid-interface (ALI) culture were examined via immunocytochemistry.

Initial studies with BEAS-2Bs eliminated HA-PEG due to no cell attachment. Bulk degradation studies revealed CC and SF retaining up to 80% of their mass by week 5. SF demonstrated the greatest Young’s modulus (23.6 ± 5.3 MPa), followed by CC (3.7 ± 0.9 MPa). BEAS-2Bs formed focal adhesions on Collagen I controls by 2h, SF by 6h, and CC by 24h, while HTECs formed focal adhesions on Collagen I and SF by 2h and by 24h on CC. Compared to organized tight-junctions and ciliated cell formation on Collagen I controls by day 14 of ALI, SF demonstrated delayed maturation by day 45 of ALI. Due to exceptional attachment (2h for BEAS-2B and HTEC) and differentiation (day 14 of ALI) capabilities of the softer CVM, a composite SF-CVM material was developed to achieve both mechanical and differentiation benefits. SF-CVM demonstrated organized tight-junctions and individual cilia forming by day 14 of ALI, and multi-ciliated cell development by day 21 of ALI.

Development of an epithelial graft that provides optimal mechanical and differentiation capabilities is essential for human tracheal regeneration. This study will have significant clinical implications for airway surgeries and viable tracheal transplants in future.