Mouse Model of Tracheal Replacement With Electrospun Nanofiber Scaffolds

Abstract

Objectives: The clinical experience with tissue-engineered tracheal grafts (TETGs) has been fraught with graft stenosis and delayed epithelialization. A mouse model of orthotopic replacement that recapitulates the clinical findings would facilitate the study of the cellular and molecular mechanisms underlying graft stenosis. Methods: Electrospun nanofiber tracheal scaffolds were created using nonresorbable (polyethylene terephthalate + polyurethane) and co-electrospun resorbable (polylactide-co-caprolactone/polyglycolic acid) polymers (n = 10/group). Biomechanical testing was performed to compare load displacement of nanofiber scaffolds to native mouse tracheas. Mice underwent orthotopic tracheal replacement with syngeneic grafts (n = 5) and nonresorbable (n = 10) and resorbable (n = 10) scaffolds. Tissue at the anastomosis was evaluated using hematoxylin and eosin (H&E), K5+ basal cells were evaluated with the help of immunofluorescence testing, and cellular infiltration of the scaffold was quantified. Micro computed tomography was performed to assess graft patency and correlate radiographic and histologic findings with respiratory symptoms. Results: Synthetic scaffolds were supraphysiologic in compression tests compared to native mouse trachea (P < .0001). Nonresorbable scaffolds were stiffer than resorbable scaffolds (P = .0004). Eighty percent of syngeneic recipients survived to the study endpoint of 60 days postoperatively. Mean survival with nonresorbable scaffolds was 11.40 ± 7.31 days and 6.70 ± 3.95 days with resorbable scaffolds (P = .095). Stenosis manifested with tissue overgrowth in nonresorbable scaffolds and malacia in resorbable scaffolds. Quantification of scaffold cellular infiltration correlated with length of survival in resorbable scaffolds (R2 = 0.95, P = .0051). Micro computed tomography demonstrated the development of graft stenosis at the distal anastomosis on day 5 and progressed until euthanasia was performed on day 11. Conclusion: Graft stenosis seen in orthotopic tracheal replacement with synthetic tracheal scaffolds can be modeled in mice. The wide array of lineage tracing and transgenic mouse models available will permit future investigation of the cellular and molecular mechanisms underlying TETG stenosis.

Applications of Electrospinning for Tissue Engineering in Otolaryngology

“…1 TETGs that are supraphysiologic in compression tests compared to native in vivo models may confer higher rates of graft stenosis; non-resorbable grafts may also contribute to more frequent stenosis (Table 1). 10,11…”

“…Sixteen studies were isolated pertaining to the application of electrospun nanofibers as tissue engineered tracheal grafts (TETG) for reconstruction of long segment tracheal defects. 1,[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] Although the specifics of scaffold design and fabrication vary for each study, Clarke and colleagues describe a representative process: First a custom mandrel was created from stainless steel with dimensions matching those obtained from native juvenile sheep tracheas. 16 Once the desired polymer blend was selected, this solution was electrospun using a custom designed apparatus comprised of 20 gauge blunt tip needles and a high voltage direct current (DC) power supply (Figure 3).…”

Tissue engineering applications in otolaryngology—The state of translation

“…Zhao et al demonstrated graft tensile and compressive strength greater than that of native trachea when using a seeded and subsequently decellularized stent with the scaffold comprised of polyglycolic acid and metal 92 . Dharmadhikari et al compared nonresorbable and resorbable scaffolds and found that both scaffolds held greater tensile strength than native trachea with nonresorbable scaffold being stiffer than resorbable 93 . However, both scaffold types were complicated by stenosis when implanted in mice with resorbable scaffolds demonstrating tracheomalacia and nonresorbable showing tissue overgrowth.…”

Regeneration of partially decellularized tracheal scaffolds in a mouse model of orthotopic tracheal replacement

“…Graft patency was assessed in vivo with the Trifoil eXplore Locus RS 80 micro-computed tomography (microCT). 6 Live animal scanning was performed on post-operative day (POD) 0; animals were positioned prone following induction of inhalational anesthesia (1%−3% isoflurane in room air at 1–3 L/min). Terminal scans were performed at humane and experimental endpoints with similar positioning.…”

“… 5 Allograft outcomes have been limited due to graft immunogenicity and synthetic tracheal scaffolds have exhibited poor epithelialization and neovascularization. 6 , 7 Initially, decellularization was geared toward removal of all native cellular material, thus limiting immunogenicity while preserving a natural scaffold composed of extracellular matrix (ECM). 8 , 9 Decellularized trachea have exhibited regeneration in pre-clinical models, however their clinical applications have remained limited.…”