1. Introduction
Long tracheal injury or stenosis have always been a difficult problem to be solved in surgical reconstruction.[1, 2] When the resections of the diseased tissue or stenosis and end-to-end anastomosis can not achieve the clinical curative effects, tissue-engineered trachea provides a new inspiration for tracheal replacement therapy.[3] Scaffolds, seed cells and growth factors are the three elements of tissue engineering. Scaffolds provide the guarantee for the adhesion and growth of cells and the role play of cytokines.[4] Therefore, appropriate tracheal scaffolds are the key to the successful implementation of tissue-engineered trachea, and the selection of scaffold materials has become the important part.[5] Autologous tissue has good vascularization and does not need immunosuppressive treatment, but its long-term application is restricted by large surgical trauma, limited scope of use and lack of epithelialization. After allogeneic transplantation, there are serious immunological rejection and lack of biological function.[6, 7] With the deepening of the interdisciplinary concept, synthetic polymer materials have been continuously developed and applied. Among them, PCL occupies a place in the field of scaffold materials for its slow rate of biodegradation and perfect biomechanical properties.[8] In previous study, we have successfully manufactured tracheal graft using PCL as the material by 3D printing technology. Biomechanical and biocompatible test proved that 3D printed tracheal graft was equipped with favorable cellular biocompatibility and biomechanical properties.[9] However, the surface hydrophobicity of PCL greatly affects cell adhesion. Therefore, we performed nano-silicon dioxide surface modification to make the surface smoother and significantly improve the cytotropism. In addition, the porous structure of 200 μm made it more conducive to cell adhesion and proliferation.[10] Even so, PCL scaffolds are still difficult to load seed cells and cytokines, and thus cannot achieve subsequent cartilaginification, vascularization and epithelization.
In recent years, hydrogels have been widely used as carriers of cells and growth factors, and their 3D network structure is conducive to the transport of seed cells, growth factors, nutrients and the discharge of metabolic wastes.[11] Silk fibroin (SF) is a natural biomaterial with good biocompatibility, excellent mechanical properties, and tunable degradability.[12, 13]Silk Fibroin Methacryloyl is the product of SF, which was modified by methylacrylylation. Its rapid solubilization in water allows SilMA to be photocurable as a hydrogel. KGN is a kind of small-molecule drug, which was discovered in 2012 for the first time.[14] It could selectively stimulate chondrogenic differentiation of endogenous bone marrow mesenchymal stem cells.[15]
Here, the purpose of this study was to prepare 3D printed hybrid tracheal graft fabricated by PCL coated with SilMA, in which BMSCs, KGN and epithelia were co-cultured, and to select the appropriate concentration. So that, the effect on cell behavior can be explored. BMSCs were isolated from tibial plateau and epithelia were cultured from autologous tracheal mucosal epithelium, which was extracted by biopsy forceps in endoscope. Biocompatibility and mechanical properties were evaluated by in vitro experiments. What’s more, the role of epithelization and cartilaginization of this hybrid tracheal graft were evaluated via in vivo window-shape defect repair(Figure 1) . This study provides the theoretic and experimental basis for further research and practice in tracheal reconstruction.