The effect of detergents on the basement membrane complex of a biologic scaffold material.
The basement membrane complex (BMC) is a critical component of the extracellular matrix (ECM) that supports and facilitates the growth of cells. This study investigates four detergents commonly used in the process of tissue decellularization and their effect upon the BMC. The BMC of porcine urinary bladder was subjected to 3% Triton-X 100, 8mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4% sodium deoxycholate or 1% sodium dodecyl sulfate (SDS) for 24h. The BMC structure for each treatment group was assessed by immunolabeling, scanning electron microscopy (SEM) and second harmonic generation (SHG) imaging of the fiber network. The composition was assessed by quantification of dsDNA, glycosaminoglycans (GAG) and collagen content. The results showed that collagen fibers within samples treated with 1% SDS and 8mM CHAPS were denatured, and the ECM contained fewer GAG compared with samples treated with 3% Triton X-100 or 4% sodium deoxycholate. Human microvascular endothelial cells (HMEC) were seeded onto each BMC and cultured for 7 days. Cell-ECM interactions were investigated by immunolabeling for integrin β-1, SEM imaging and semi-quantitative assessment of cellular infiltration, phenotype and confluence. HMEC cultured on a BMC treated with 3% Triton X-100 were more confluent and had a normal phenotype compared with HMEC cultured on a BMC treated with 4% sodium deoxycholate, 8mM CHAPS and 1% SDS. Both 8mM CHAPS and 1% SDS damaged the BMC to the extent that seeded HMEC were able to infiltrate the damaged sub-basement membrane tissue, showed decreased confluence and an atypical phenotype. The choice of detergents used for tissue decellularization can have a marked effect upon the integrity of the BMC of the resultant bioscaffold.
Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15219, USA; McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA.
D M
DM
Faulk
C A
CA
Carruthers
H J
HJ
Warner
C R
CR
Kramer
J E
JE
Reing
L
L
Zhang
A
A
D'Amore
S F
SF
Badylak
D000818:Animals; D001485:Basement Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D003094:Collagen / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D020287:In Situ Nick-End Labeling; D019012:Integrin beta1 / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D055806:Microvessels / Q000166:cytology; D013194:Staining and Labeling; D034421:Sus scrofa; D054457:Tissue Scaffolds / Q000737:chemistry*
D003902:Detergents; D006025:Glycosaminoglycans; D019012:Integrin beta1; D019394:Ki-67 Antigen; C013786:A73025; D003094:Collagen; D004247:DNA
Biologic scaffold; Decellularization; Extracellular matrix; Organ engineering; Re-endothelization