Abstract: BACKGROUND: For the replacement treatment of long-segment tracheal defects, although tissue engineering research has made some progress in recent years, it is still not perfect, and one of the biggest difficulties is that the hemodynamic reconstruction of the tracheal replacement cannot be achieved rapidly. 
OBJECTIVE: To preliminarily explore the potential of polycaprolactone scaffolds modified with exosome-loaded hydrogels to construct a rapidly vascularized tracheal substitute. 
METHODS: Exosomes were extracted from bone marrow mesenchymal stem cells of SD rats. After preparation of hyaluronic acid methacrylate solution, the exosome solution was mixed with hyaluronic acid methacrylate solution at a volume ratio of 1:1. Hyaluronic acid methacrylate hydrogels loaded with exosomes were prepared under ultraviolet irradiation for 5 minutes. The degradation of exosome-unloaded hydrogels and the controlled release of exosome-loaded hydrogels were detected. Polycaprolactone scaffolds were prepared by 3D printing. The pure hyaluronic acid methacrylate solution and the exosome-loaded hyaluronic acid methacrylate solution were respectively added to the surface of the scaffold. Hydrogel-modified scaffolds and exosome-modified scaffolds were obtained after ultraviolet irradiation. Thirty SD rats were randomly divided into three groups with 10 rats in each group and subcutaneously implanted with simple scaffolds, hydrogel-modified scaffolds and exosome-modified scaffolds, respectively. At 30 days after surgery, the scaffolds and surrounding tissues of each group were removed. Neovascularization was observed by hematoxylin-eosin staining and Masson staining and the expression of CD31 was detected by immunofluorescence. 
RESULTS AND CONCLUSION: (1) As time went by, the hydrogel degraded gradually, and the exosomes enclosed in the hydrogel were gradually released, which could be sustained for more than 30 days. The exosome release rate was faster than the degradation rate of the hydrogel itself, and nearly 20% of the exosomes were still not released after 30 days of soaking. (2) Under a scanning electron microscope, the surface of the simple polycaprolactone scaffold was rough. After hydrogel modification, a layer of gel was covered between the pores of the scaffold, and the scaffold surface became smooth and dense. (3) After 30 days of subcutaneous embedding, hematoxylin-eosin staining and Masson staining showed that more neovascularization was observed inside the scaffolds of the exosome-modified scaffold group compared with the hydrogel-modified scaffold group. The hydrogels on the scaffolds of the two groups were not completely degraded. Immunofluorescence staining showed that CD31 expression in the exosome-modified scaffold group was higher than that in the hydrogel-modified scaffold group (P < 0.000 1). (4) These results indicate that hyaluronic acid methacrylate hydrogels can be used as controlled-release carriers for exosomes. The 3D-printed polycaprolactone scaffold modified by hyaluronic acid methacrylate hydrogel loaded with exosomes has good biocompatibility and has the potential to promote the formation of neovascularization. 

Key words: tracheal reconstruction, exosome, polycaprolactone, tissue engineering, 3D printing, vascularization, hydrogel