AVS 62nd International Symposium & Exhibition
    Biomaterial Interfaces Monday Sessions
       Session BI+AS-MoA

Paper BI+AS-MoA3
Molecular-Level Surface Analysis Demonstrates the Impact of Detergent Selection on Decellularized Tissues

Monday, October 19, 2015, 3:00 pm, Room 211D

Session: Characterization of Biological and Biomaterials Surfaces (2)
Presenter: Adam Taylor, University of Washington
Authors: A.J. Taylor, University of Washington
L.J. White, University of Nottingham, UK
D.M. Faulk, University of Pittsburgh
L.T. Saldin, University of Pittsburgh
D.G. Castner, University of Washington
S.F. Badylak, University of Pittsburgh
B.D. Ratner, University of Washington
Correspondent: Click to Email
Decellularized matrix scaffolds may be prepared through a range of techniques. Detergents are frequently used in decellularization protocols due to their ability to solubilize cell membranes and dissociate DNA from proteins. Whilst removal of cellular material is regularly assessed, the impact of detergent selection on extracellular matrix (ECM) structure and composition is less commonly investigated. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful surface analysis technique to probe biological structures with high mass resolution and surface specificity, and has previously been used to distinguish decellularized ECM by anatomical location or culture conditions. The objective of this study was to utilize ToF-SIMS to investigate the influence of detergent selection upon a representative decellularized tissue, specifically the basement membrane complex (BMC) of porcine urinary bladder matrix (UBM) prepared by treatment with 1% SDS, 4% deoxycholate, 8 mM CHAPS or 3% Triton X-100 for 24 hours.

Principal components analysis (PCA) revealed spectral differences between treatment groups. High mass peaks associated with specific detergent fragments were observed on the scaffolds exposed to SDS and deoxycholate. Peaks indicative of phospholipid membranes were observed in all samples, but to a greater extent with scaffolds not exposed to detergent. We further probed these data sets to investigate how detergent selection impacts proteinaceous ECM components. Using a reduced peak list of known characteristic amino acid fragments, PCA distinguished native bladder tissue from decellularized UBM and highlighted spectral differences between UBM treated with ionic vs. charge-neutral detergents. Notably, the basement membrane surface of UBM prepared with ionic detergents SDS and deoxycholate yielded less intense characteristic peaks from hydrophobic amino acids than UBM treated with charge neutral detergents CHAPS and Triton X-100. Harsher detergents may denature protein structure and break protein-protein interactions through binding of their hydrophobic tail to hydrophobic amino acid residues. Such damage is hypothesized to cause sub-optimal in vitro and in vivo responses. We further examined cell-matrix interactions of human urothelial cells seeded on the BMC of UBM, investigating how detergent exposure affected cell proliferation and permeability of the cell monolayer. An understanding of the effects of detergent exposure on the structure, composition and surface molecular functionality of decellularized scaffolds will facilitate a rational strategy for successful recellularization and subsequent positive clinical outcomes.