Histological evaluation of decellularised porcine aortic valves: matrix changes due to different decellularisation methods

Abstract

OBJECTIVE: Several decellularisation techniques have been developed to produce acellular matrix scaffolds for the purpose of tissue engineering, mostly comprising (non-)ionic detergents or enzymatic extraction methods. However, the effect of chemically induced decellularisation on the major structural and adhesion molecules as well as glycosaminoglycans, and the possible replenishment of lost compounds have escaped attention. METHODS: Porcine aortic valves were treated with two different methods: detergent Triton X-100 and enzymatic Trypsine cell extraction. (Immuno-) histochemistry was used to address changes in extracellular matrix constitution (elastin, collagen, glycosaminoglycans, chondroitin sulfate, fibronectin and laminin) and the production of extracellular matrix components by seeded endothelial cells. RESULTS: The Trypsine treated group showed a fragmentation and distortion of elastic fibers. Changes in collagen distribution were observed in both groups. An almost complete washout of glycosaminoglycans and chondroitin sulfate was observed in the Triton and Trypsin treated group, but the latter with a smaller glycosaminoglycans reduction. Both treatments resulted in a considerable washout of the adhesion molecules laminin and fibronectin. Furthermore, seeded endothelial cells were capable of synthesising laminin, fibronectin and chondroitin sulfate. CONCLUSIONS: Chemically induced decellularisation by Triton or Trypsine resulted in changes in the extracellular matrix constitution, which could lead to problems in valve functionality and cell growth and migration. Seeded endothelial cells were capable of synthesising extracellular matrix components lost by cell extraction. Further studies on tissue engineering should focus more on the effect of chemically induced cell extraction on the extracellular matrix of the remaining scaffold and the in vitro or in vivo replenishment of lost compounds.