Dynamic flow for efficient partial decellularization of tracheal grafts: A preliminary rabbit study

Abstract

Objective: Bioengineered tracheal grafts are a potential solution for the repair of long-segment tracheal defects. A recent advancement is partially decellularized tracheal grafts (PDTGs) which enable regeneration of host epithelium and retain viable donor chondrocytes for hypothesized benefits to mechanical properties. We propose a novel and tunable 3D-printed bioreactor for creating large animal PDTG that brings this technology closer to the bedside.

Methods: Conventional agitated immersion with surfactant and enzymatic activity was used to partially decellularize New Zealand white rabbit (Oryctolagus cuniculus) tracheal segments (n = 3). In parallel, tracheal segments (n = 3) were decellularized in the bioreactor with continuous extraluminal flow of medium and alternating intraluminal flow of surfactant and medium. Unprocessed tracheal segments (n = 3) were also collected as a control. The grafts were assessed using the H&E stain, tissue DNA content, live/dead assay, Masson's trichrome stain, and mechanical testing.

Results: Conventional processing required 10 h to achieve decellularization of the epithelium and submucosa with poor chondrocyte viability and mechanical strength. Using the bioreactor reduced processing time by 6 h and resulted in chondrocyte viability and mechanical strength similar to that of native trachea.

Conclusion: Large animal PDTG created using our novel 3D printed bioreactor is a promising approach to efficiently produce tracheal grafts. The bioreactor offers flexibility and adjustability favorable to creating PDTG for clinical research and use. Future research includes optimizing flow conditions and transplantation to assess post-implant regeneration and mechanical properties.

Level of evidence: NA.

Keywords: bioreactor; chondrocyte viability; regenerative medicine; tissue engineering; tracheal replacement.

FIGURE 1

Bioreactor design and setup: (A) Cross‐section of the bioreactor with mock tracheal segment. The dotted arrows represent the intraluminal and extraluminal flow paths. The pumps are represented on the right with the rectangle representing flexible fluid valving and splitting. (B) Rabbit trachea which has been affixed to the two modular end fittings of the bioreactor. (C) Fully installed bioreactor with connected tubing and media flow.

FIGURE 2

H&E staining and DNA content assay: (A–C) Representative axial H&E images of native and processed trachea. The triangles denote the epithelium while the arrows denote the cartilage. (D) DNA content assay results which demonstrate no differences between the groups.

FIGURE 3

Live/dead assay and viability quantification: (A–C) Representative axial live/dead images of native and processed trachea. (D) Quantified viability demonstrating that conventional processing results in partially decellularized tracheal grafts with chondrocyte viability lower than that of native and bioreactor‐processed trachea. (*** denotes significance with P ≤ .001).

FIGURE 4

Masson's trichrome: (A, B, D) Representative axial images of native and processed trachea stained with Masson's trichrome. (C) A representative cropped and stain‐only image of (B) processed using the AFAT algorithm. (E) Stain percentages which demonstrate no differences between the groups.

FIGURE 5

Compression testing method and results: (A) Example load‐compression graph with images demonstrating correlated tracheal occlusion. The arrow shows the datum at 50% occlusion which was used to compare between groups. (B) Force at 50% occlusion which demonstrates that conventional processing results in partially decellularized tracheal grafts that occlude at lower loads when compared to native and bioreactor‐processed trachea. (* denotes significance with P ≤ .05).