DFG-Projekt "Viskograft"

 

 

REM & Histologie



Silvia Mangold am REM der HS.R im Labor von Prof. Dr. Hammer




Vergoldete HUV-Proben beim Ladevorgang im REM


Abstract
Human umbilical vessels have been recognized as a valuable and widely available resource
for vascular tissue engineering. Whereas endothelium-denuded human umbilical
veins (HUV) have been successfully seeded with a patient-derived neoendothelium, decellularized
vessels may have additional advantages due to their lower antigenicity. The
present study investigated the effects of three different decellularization procedures on
histological, mechanical, and seeding properties of HUV. Vessels were decellularized by
detergent treatment (Triton X-100, sodium deoxycholate, IGEPAL-CA630), osmotic lysis
(3M NaCl, distilled water), and by peroxyacetic acid treatment. In all cases nuclease
treatments were required to remove residual nucleic acids. Decellularization resulted in
a partial loss of fibronectin and laminin staining in the subendothelial layer and affected
the appearance of elastic fibers. In addition to removing residual nucleic acids, nuclease
treatment weakened all stainings and substantially altered surface properties as seen in
scanning electron micrographs, indicating additional nonspecific effects. Detergent
treatment and osmotic lysis caused failure stresses to decrease significantly. Although
conditioned media prepared from decellularized HUV did not severely affect endothelial
cell growth, cells seeded on decellularized HUV did not remain viable. This may be attributed
to the partial removal of essential extracellular matrix components as well as to
changes of surface properties. Therefore, decellularized HUV appear to require additional
modifications in order to support successful cell seeding. Replacing the vessels'
endothelium only should be considered as an alternative to decellularization when creating
tissue engineered blood vessels with non-immunogenic luminal interfaces.

Keywords: vascular tissue engineering, small caliber graft, endothelium, biomechanics,
human umbilical vein


REM

Scanning electron microscopy images of HUV luminal surfaces. (A) native HUV; (B)
endothelium-denuded HUV; (C) HUV decellularized by detergents before and (D) after
nuclease treatment; (E) HUV decellularized by osmotic lysis before and (F) after nuclease
treatment; (G) HUV decellularized by PAA before and (H) after nuclease treatment. Images
are representative for 3 independent preparations per condition. Bars indicate 20 μm.

Histologie


Histological evaluation of HUV decellularization followed by nuclease treatment. See
Fig. 1 for explanations. Images are representative for 3-4 independent sample preparations.
Bar indicates 100 μm.


Veröffentlichung:

[15] Mangold, S.; Schrammel, S.; Huber, G.; Niemeyer, M.; Schmid, C.; Stangassinger, M.; Hoenicka, M.:
Evaluation of decellularized human umbilican vein (HUV) for vascular tissue engineering - comparison with endothelium-denuded HUV
Journal of Tissue Engineering, TERM-12-0055.R1, 2012