Development and In Vitro/In Vivo Comparative Characterization of Cryopreserved and Decellularized Tracheal Grafts

Abstract

Tracheal reconstruction represents a challenge when primary anastomosis is not feasible. Within this scenario, the study aim was to develop a new pig-derived decellularized trachea (DecellT) to be compared with the cryopreserved counterpart (CryoT) for a close predictive analysis. Tracheal segments underwent decellularization by a physical + enzymatic + chemical method (12 cycles); in parallel, cryopreserved samples were also prepared. Once decellularized (histology/DNA quantification), the two groups were characterized for Alpha-Gal epitopes/structural proteins (immunohistochemistry/histology/biochemical assays/second harmonic generation microscopy)/ultrastructure (Scanning Electron Microscopy (SEM))/mechanical behaviour. Cytotoxicity absence was assessed in vitro (extract-test assay/direct seeding, HM1SV40 cell line) while biocompatibility was verified in BALB/c mice, followed by histological/immunohistochemical analyses and SEM (14 days). Decellularization effectively removed Alpha-Gal epitopes; cartilage histoarchitecture was retained in both groups, showing chondrocytes only in the CryoT. Cryopreservation maintained few respiratory epithelium sparse cilia, not detectable in DecellT. Focusing on ECM, preserved structural/ultrastructural organization and collagen content were observed in the cartilage of both; conversely, the GAGs were significantly reduced in DecellT, as confirmed by mechanical study results. No cytotoxicity was highlighted by CryoT/DecellT in vitro, as they were also corroborated by a biocompatibility assay. Despite some limitations (cells presence/GAGs reduction), CryoT/DecellT are both appealing options, which warrant further investigation in comparative in vivo studies.

The suprapatellar fat pad: A histotopographic comparative study

“…For easier evaluation, the white and black colors were inverted. On the processed images, the white‐colored areas corresponding to collagen/elastic fibers were selected and automatically measured (Barbon, Stocco, et al., 2022; Stocco et al., 2023). Results were expressed as arbitrary unit (AU).…”

“…Thus, once identified, selected, and set, the most proper hue, saturation, and brightness ranges' thresholds for the red‐orange (collagen type I) and green‐yellow (collagen type III) areas (0–39, 210–255; 78–255; 10–255 and 39–120; 78–255; 10–256, respectively) and their amounts were calculated to better describe collagen type (I/III) prevalence within the samples. Both the septa (previously selected manually) and the area surrounding the adipocytes (the whole image was here considered) were analyzed and the results were expressed as arbitrary unit (AU) (Stocco et al., 2023).…”

Tissue‐engineered tracheal implants: Advancements, challenges, and clinical considerations

“…29 Subsequent explorations involved diverse grafting materials, such as autogenous skin, 30 fascial tissue, 31 and costal cartilage, 32 along with the introduction of solid materials in prostheses. 33 Tubular tracheal substitutes have been the focus of extensive investigation, employing various strategies such as cadaveric tissue flaps, 34 implanted bioprostheses, 35 tubes derived from intestinal tissue 36,37 and different types of aortic grafts. 38 The outcomes, successes, and limita- 41,42 Nevertheless, the explored techniques exhibit substantial promise and have the potential to establish the gold standard in tissue-engineered tracheal constructs.…”

“…The initial human application of primary anastomosis took place in 1886, marking a significant milestone 29 . Subsequent explorations involved diverse grafting materials, such as autogenous skin, 30 fascial tissue, 31 and costal cartilage, 32 along with the introduction of solid materials in prostheses 33 . Tubular tracheal substitutes have been the focus of extensive investigation, employing various strategies such as cadaveric tissue flaps, 34 implanted bioprostheses, 35 tubes derived from intestinal tissue 36,37 and different types of aortic grafts 38 .…”