Development of Advanced Indirect Three Dimensional Printing Technique and Its Application to Tracheal Tissue Engineering

Abstract

Reconstruction of a tracheal defect remains as one of the current challenges in the field of tissue engineering. Although tracheal disease is rare, there is an apparent need to reconstruct tracheal defects because of its extremely high mortality rate. To date, diverse approaches to reconstruct tracheal defects using artificial graft have been reported; but still, tracheal graft with satisfactory results has not developed yet. The trachea has adequate flexibility and mobility to ensure its stability in the body even when the neck is rotated, extended, and flexed. In this light, artificial graft for tracheal reconstruction should be enabled to exhibit relevant mechanical behavior similar to native trachea. In this research, a novel tissue-engineered tracheal graft was developed with a consideration of the actual mechanical behavior of the native trachea and its validation was performed for long-segment circumferential tracheal reconstruction. In the first step, an advanced indirect 3D printing technique was developed as a reliable method for creating the tracheal graft with intact structural integrity. Projection based microstreolithography (pMSTL) system was built and employed to print a sacrificial mold and injection molding system (IMS) was newly developed in order to perform a thermal molding process, resulting in overcoming inherent limitations in previous techniques based on solvent-based molding process. In a next step, bellows scaffold which is capable of exhibiting relevant mechanical behavior similar to native trachea was developed based on the native-trachea mimetic design. Its mechanical behavior under three-point bending and radial compression was analyzed using FEM analysis. Then, human turbinate mesenchymal stromal cell (hTMSC) sheets were applied to facilitate tracheal epithelial regeneration. hTMSC sheets were prepared using a temperature-responsive culture dishes and their differentiation capacity into the tracheal epithelium was assessed in vitro. Tracheal epithelial regeneration ability of hTMSC sheets with bellows scaffold was also evaluated using a non-circumferential tracheal defect in a rabbit model. Finally, the performance of the tissue-engineered tracheal graft was demonstrated by reconstruction of long-segment circumferential tracheal defect. Prior to in vivo test using tissue-engineered tracheal graft, immunosuppressive effect in a rabbit model was investigated for safe application of hTMSC sheets without adverse side effect. Bellows scaffold, with a critical size of the tracheal length in infants, was then fabricated via advanced indirect 3D printing technique and the tissue-engineered tracheal graft was successfully prepared from bellows scaffold reinforced with silicon rings, mucosal decellularized extracellular matrix (mdECM) hydrogel, and hTMSC sheets. In vivo test, it was successfully demonstrated that pseudostratified columnar epithelium was completely regenerated in the luminal surface of the tissue-engineered tracheal graft within 2 months after the implantation.