Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide

Abstract

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 cellremoving detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propa nesulfonate (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. Statement of Significance 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.

Graft-specific immune tolerance is determined by residual antigenicity of xenogeneic extracellular matrix scaffolds

“…Previous reports have demonstrated that disruption of ECM components following decellularization/antigen removal processes has potential to result in innate immune system activation [6,26,32,41], resulting in fibrous encapsulation and foreign body response [18,33,42]. In the current study, GFBP implants resulted in marked innate immune activation and resultant fibrous encapsulation.…”

“…This distinction suggests that the innate immune response is tolerant to minute deviations in ECM protein organization provided proteins are not denatured. Other literature also observed this phenomenon when examining the innate immune response to collagen denaturation [32,41,47]. For example, harsh decellularization methods (e.g., SDS) have been previously documented to result in significant ECM damage.…”

“…However, ionic detergents, such as sodium dodecyl sulfate (SDS), have been shown to have surprisingly poor performance in solubilization and removal of lipophilic antigens. This results in persistence of both known (e.g., MHC I) and unknown (i.e., 70% residual antigenicity remaining) lipophilic antigens [3,13,32]. Furthermore, studies have shown SDS to severely denature vital ECM proteins and be toxic to repopulating cells, stimulating recipient innate immune mediated foreign body response [3,10,13,33–35].…”

Dietary Advanced Glycation End-Product Consumption Leads to Mechanical Stiffening of Murine Intervertebral Discs

“…This study demonstrated increased CHP staining, as a direct measure of damage to the triple helical structure of collagen molecules, in the H-AGE female group, which may also account for the reduced SHG intensity. Alterations in fibril packing can increase the susceptibility to enzymatic digestion (McBride et al, 1997) and collagen degradation, which in turn can contribute to the loss of collagen SHG intensity (Hwang et al, 2017). It has been proposed that AGE crosslinking can lead to mechanically induced breakdown of the collagen triple helical structure by causing forces, transmitted through the intermolecular AGE crosslinks, to accelerate enzyme degradation (Bourne et al, 2014).…”

“…PMMA embedded mid-sagittal lumbar sections were stained using Collagen Hybridizing Peptide-biotin conjugate (B-CHP; BIO300, 3Helix Inc; Salt Lake City, UT, USA) to detect the presence of collagen damage as described by (Hwang et al, 2017, Zitnay et al, 2017. This procedure includes the creation of a 2 µM CHP solution, heating of the solution at 80°C for 5 minutes to monomerize the peptides, quenching in an ice bath for 30 seconds, and application of the solution to the tissue.…”