Long-Term Chondrocyte Retention in Partially Decellularized Tracheal Grafts.

OBJECTIVE: Decellularized tracheal grafts possess the biological cues necessary for tissue regeneration. However, conventional decellularization approaches to target the removal of all cell populations including chondrocytes lead to a loss of mechanical support. We have created a partially decellularized tracheal graft (PDTG) that preserves donor chondrocytes and the mechanical properties of the trachea. In this study, we measured PDTG chondrocyte retention with a murine microsurgical model. STUDY DESIGN: Murine in vivo time-point study. SETTING: Research Institute affiliated with Tertiary Pediatric Hospital. METHODS: PDTG was created using a sodium dodecyl sulfate protocol. Partially decellularized and syngeneic grafts were orthotopically implanted into female C57BL/6J mice. Grafts were recovered at 1, 3, and 6 months postimplant. Pre- and postimplant grafts were processed and analyzed via quantitative immunofluorescence. Chondrocytes (SOX9+, DAPI+) present in the host and graft cartilage was evaluated using ImageJ. RESULTS: Partial decellularization resulted in the maintenance of gross tracheal architecture with the removal of epithelial and submucosal structures on histology. All grafts demonstrated SOX9+ chondrocytes throughout the study time points. Chondrocytes in PDTG were lower at 6 months compared to preimplant and syngeneic controls. CONCLUSION: PDTG retained donor graft chondrocytes at all time points. However, PDTG exhibits a reduction in chondrocytes at 6 months. The impact of these histologic changes on cartilage extracellular matrix regeneration and repair remains unclear.

Optimization of Chondrocyte Viability in Partially Decellularized Tracheal Grafts.

OBJECTIVE: Advancements in tissue-engineered tracheal replacement (TETR) show promise for the use of partially decellularized tracheal grafts (PDTG) to address critical gaps in airway management and reconstruction. In this study, aiming to leverage the immunoprivileged nature of cartilage to preserve tracheal biomechanics, we optimize PDTG for retention of native chondrocytes. STUDY DESIGN: Comparison in vivo murine study. SETTING: Research Institute affiliated with Tertiary Pediatric Hospital. METHODS: PDTG were created per a shortened decellularization protocol using sodium dodecyl sulfate, then biobanked via cryopreservation technique. Decellularization efficiency was characterized by DNA assay and histology. Viability and apoptosis of chondrocytes in preimplanted PDTG and biobanked native trachea (control) was assessed with live/dead and apoptosis assays. PDTG (N = 5) and native trachea (N = 6) were orthotopically implanted in syngeneic recipients for 1-month. At the endpoint, microcomputed tomography (micro-CT) was employed to interrogate graft patency and radiodensity in vivo. Vascularization and epithelialization were qualitatively analyzed using histology images following explant. RESULTS: PDTG exhibited complete decellularization of all extra-cartilaginous cells and reduced DNA content compared to control. Chondrocyte viability and nonapoptotic cell populations were improved utilizing biobanking and shorter decellularization time. All grafts remained patent. Evaluation of graft radiodensity at 1 month revealed elevation of Hounsfield units in both PDTG and native compared to host, with PDTG showing higher radiodensity than native. PDTG supported complete epithelialization and functional reendothelialization 1-month postimplantation. CONCLUSION: Optimizing PDTG chondrocyte viability is a key component to successful tracheal replacement. Ongoing research seeks to evaluate the acute and chronic immunogenicity of PDTG.