Chondrogenesis in primitive tracheal neocartilage: insights from 3D-printed silicone grafts in a large-scale animal model.

OBJECTIVES: Tracheal cartilage has limited regenerative potential in adults, posing a significant challenge for tracheal repair. Here, we observed marked neocartilage growth in a porcine model after transplanting it with a three-dimensional (3D)-printed silicone tracheal graft. METHODS: Virtual silicone tracheal grafts of 2 cm in length were first generated using a 3D printer, and simulated a 3-month-old porcine trachea based on data from physical stress tests. After a segmental resection, a graft underwent end-to-end anastomosis at both the proximal and distal tracheal sections. Neotissue samples were later examined macroscopically, and their histologic properties were assessed based on results from the following: protein expression using H&E staining, alcian blue and safranin O/fast green staining for glycosaminoglycans (GAGs), and immunohistochemistry (IHC) assays for Sox9, type II collagen, aggrecan, and proliferating cell nuclear antigen (PCNA). RESULTS: These primitive cartilages displayed chondrogenesis with an initial surplus of chondrocytes, evolving into a mature, stable state with cartilage corrosion facilitated by interim perichondrium-derived chondro-modulators, like perichondrial papillae (PPs), preresorptive layers (PRLs), and vascular canals (VCs). Results of alcian blue staining revealed the removal of matrix degradation products, specifically in the vicinity of the mucosal and submucosal glands. CONCLUSIONS: Chondrogenesis underwent an initial burst of growth together with the perichondrium, constituting a gradual consolidation process. Such a consolidation was supported by the emission of matrix degradation products in the mucosa and submucosa. To summarize our results, we propose a four-stage scheme to characterize the regenerative chondrogenesis of tracheal neocartilage.