Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds

Abstract

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3–8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.

Residual sodium dodecyl sulfate in decellularized muscle matrices leads to fibroblast activation in vitro and foreign body response in vivo

“…Scanning electron microscopy images shown in Figure a demonstrate that decellularization of muscle tissue in 0.25% SDS under 20 inHg (508 mmHg) of negative pressure during processing did not alter tissue morphology compared to matrices processed with the same detergent treatment under atmospheric pressure. This result is consistent with recently published work that showed intact tissue morphology in trachea decellularized under 1 Torr negative pressure, a stronger vacuum than used in our study (Butler et al, ). Athough we did not specifically test the effects of negative pressure on increasing efficiency of decellularization, a recent study using an SDS‐based method to decellularize trachea under 200 mmHg pressure compared to atmospheric pressure suggested they could reduce the treatment time from 72 to 48 hr while obtaining high decellularization efficiencies, which corroborates our anecdotal observation that negative pressure speeds decellularization (Xu et al, ).…”

Decellularized Cartilage Directs Chondrogenic Differentiation: Creation of a Fracture Callus Mimetic

“…Recent studies applying vacuum-assisted decellularisation demonstrate increased decellularisation efficiency, especially with respect to complex organs that include dense tissues i.e. trachea and larynx [33,46]. The enhanced cellular clearance reduces the need for harsh physical or enzymatic treatments that may contribute to the loss of bioactive molecules from the ECM [33,46].…”

Biomedical grafts for tracheal tissue repairing and regeneration “Tracheal tissue engineering: an overview”

“…Xu et al () found that the microporous decellularized matrix fabricated by laser micropore technique (LMT) results in mild loss of ECM, which provides homogenous cell distribution of the matrix and good mechanical stability of graft under in situ condition. Butler et al () also reported that the vacuum‐assisted decellularization of tracheal graft results better graft for clinical application. Clinical study of acellular graft was done for the tracheal tissue reconstruction (Elliott et al, ; Etienne et al, ; Li et al, ; Martinod et al, ).…”