Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.
Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold's regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cell-removing detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology.



Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology.
Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA.
Jeongmin
J
Hwang
Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA.
Boi Hoa
BH
San
McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA; Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Neill J
NJ
Turner
School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK.
Lisa J
LJ
White
McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA.
Denver M
DM
Faulk
McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA; Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA; Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15219, USA.
Stephen F
SF
Badylak
Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA. Electronic address: yang.d.li@utah.edu.
Yang
Y
Li
Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA; Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
S Michael
SM
Yu
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002474:Cell-Free System / Q000737:chemistry*; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry* / Q000648:ultrastructure; D003902:Detergents / Q000737:chemistry*; D005109:Extracellular Matrix / Q000737:chemistry*; D008853:Microscopy / Q000379:methods; D010455:Peptides / Q000737:chemistry*; D011489:Protein Denaturation; D013194:Staining and Labeling; D013552:Swine; D001743:Urinary Bladder / Q000033:anatomy & histology / Q000737:chemistry
D003902:Detergents; D010455:Peptides; D003094:Collagen
Biologic scaffolds; Detergent; Extracellular matrix; Tissue engineering; Triple helix