Comparison of aortic valve allograft decellularization techniques in the rat

Abstract

Rodent models have been essential to understanding the immune-mediated failure of aortic valve allografts (AVAs). Decellularization has been proposed to reduce the immunogenicity of AVAs. The objective of this study was to determine the most effective method to decellularize AVAs for use in a rat model. Three different decellularization techniques were compared in Lewis aortic valves. Detergent decellularization involved a series of hypotonic and hypertonic Tris buffers at 4 degrees C for 48 h/buffer containing 0.5% Triton X-100 followed by a 72 h washout in phosphate-buffered saline. Osmotic decellularization was performed in similar manner to the detergent-based technique except without the addition of Triton X-100. Enzymatic decellularization consisted of trypsin/EDTA at 37 degrees C for 48 h. Assessment was performed with light microscopy (H&E, Movat's pentachrome), immunohistochemistry for residual cellular elements, and hydroxyproline assays. Detergent-based methodology effected near-complete decellularization of both the leaflets and aortic wall in addition to preservation of the extracellular matrix (ECM). Osmotic lysis was associated with preservation of ECM and moderate decellularization. Enzymatic decellularization resulted in complete decellularization but extensive degeneration and fragmentation of the ECM. When implanted into the infrarenal aorta of allogeneic rats for 1 week, valves decellularized with detergent-based and osmotic methodology failed to stimulate an allogeneic immune response as evidenced by an absence of T cell infiltrates. Osmotic lysis protocols with low dose detergent appear to be most effective at both removing antigenic cellular elements and preserving ECM.

Preparation of Ex Vivo–Based Biomaterials Using Convective Flow Decellularization

“…9,15,[23][24][25][26][27][28] Human saphenous veins have been decellularized by placing the specimens in a 378C sodium dodecyl sulfate (SDS) solution …”

“…29 Following similar technologies, others have produced acellular liver stroma, 8 rat aortic valve, 24,26 and iliac artery. 30 Although the molecular details are not clear, some methods are more effective than others.…”

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“…23 Although most decellularization methods are successful in reducing tissue cellularity, some methods also alter tissue integrity. 22,24,25 Detergent and enzyme decellularization methods have been reported to be associated with ECM disruption and degraded basement membranes. 26,27 We investigated 3 different protocols to decellularize heart valve samples using collagenase enzyme.…”

Chemical Decellularization Methods and Its Effects on Extracellular Matrix

“…These chemical's compositions destruct cell connections to collagen and fibronectin by separating ions such as Ca 2+ and Mg 2 (97-100). Chelating agents are usually used in combination with enzymes such as trypsin (9,52,(101)(102)(103) or detergents (9,101) and do not have any application on its own (104). EDTA in the long term reduces the mechanical properties of the scaffold (105).…”