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

Date Published: April 8, 2017

Publisher:

Author(s): Jeongmin Hwang, Boi Hoa San, Neill J. Turner, Lisa J. White, Denver M. Faulk, Stephen F. Badylak, Yang Li, S. Michael Yu.

http://doi.org/10.1016/j.actbio.2017.01.079

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.

Partial Text

Extracellular matrix (ECM) obtained by the decellularization of tissues has become an important biomaterial for tissue engineering and regenerative medicine [1]. Dozens of products derived from decellularized tissues, commonly known as biologic scaffolds, are currently used in clinical practice [1] for applications in wound care [2,3], pericardial reconstruction [4], and heart valve replacement [5], among others. By presenting a combination of structural and biological factors, such as the three-dimensional (3D) ultrastructure, mechanical integrity and a specific ECM composition, these acellular biologic scaffolds can provide a near-native and complex microenvironment for cell growth and tissue development, which is difficult to recapitulate with synthetic materials or a single ECM component. The ECM can also be partially digested with pepsin, and reconstituted in situ to form a hydrogel [6,7], which retains numerous biochemical constituents found in native tissues, such as growth factors and glycosaminoglycan. When the hydrogel forms in the damaged tissue, these bioactive cues can promote a constructive host remodeling response while curbing inflammation and scarring [6]. One example is the use of decellularized myocardial matrix hydrogels as a post-myocardial infarction biomaterial therapy [8]. This new therapy has produced encouraging preclinical results [9,10] and is currently under a clinical trial [6]. In recent years, improvement in whole organ decellularization techniques has enabled 3D organ scaffolds, which preserve the native tissue architecture including vascular networks [11,12]. These decellularized scaffolds can be repopulated with selected cell types in vitro to regenerate functional organs. So far, a variety of organs, including heart [13], kidney [14], liver [15], lung [16,17], limb [18], and pancreas [19], have been created from decellularized whole organs, and their short-term functions have been demonstrated after transplantation in vivo. In light of critical shortage of organ donors, this technological breakthrough provides hopes of transplanting engineered animal organs to patients with end-stage organ failure.

In this study, CHP was used to investigate the denaturation of triple helical collagen molecules in ECM-based tissue scaffolds after they were decellularized by common detergent treatment protocols. The reduced SHG signals (Fig. 3) and non-native fibril morphology displayed under TEM (Fig. 5) demonstrated that all four detergents (SDS, CHAPS, SD, and Triton X-100) can alter the structure of collagen fibrils. Moreover, the CHP staining (Figs. 1, 2, and 7), CD spectra (Fig. 4), and TEM images of CHP-NP binding (Fig. 6) showed that SDS and Triton X-100 are able to unravel the triple helix of a collagen molecule, while CHAPS and SD are unable to do so, even when their concentrations are doubled from common decellularization conditions (Fig. S3). The results were consistent in all three forms of collagen tested: solubilized type I collagen molecules, reconstituted and self-assembled type I collagen fibrils, and native collagen fibers in two different types of tissue (porcine ligament and UBM). We recently reported an in depth study of the effects of detergents on the structure and composition of the UBMECM, using immunohistochemistry, SEM and SHG imaging [25]. In the report, the multiphoton images of the CHAPS- and SDS-treated matrices showed similar levels of reduction of SHG signals, and their SEM images both showed amorphous structures lacking distinct fibers [25], suggesting that the two detergents had similar collagen denaturing properties. In contrast, the CHP hybridization study (Fig. 7) revealed that the two detergents have very different denaturing effects at the molecular level: the UBM matrix decellularized by 1% SDS contains substantial amounts of denatured collagen, whereas the UBM decellularized by CHAPS contained almost no denatured collagen. Such results are now self-evident because the denatured collagen molecules in a decellularized tissue can be directly visualized by a specific positive signal.

We characterized the molecular denaturation of collagen in tissues as a result of common detergent treatment (SDS, CHAPS, SD and Triton X-100). The study was made possible by a novel collagen hybridizing peptide that specifically anneals to the unfolded collagen chains through triple helix formation. By staining tissue samples with fluorescently labeled CHP and subsequent quantitative image analysis, we showed that while all four detergents alter the fibrillar structure of collagen, only SDS and Triton X-100 have the propensity to denature the collagen molecules. The results were consistent in all test samples: self-assembled type I collagen fibrils, decellularized porcine ligament, and urinary bladder matrix. The results were also in agreement with other characterization methods, including SHG, CD and TEM. The triple helix is the most fundamental structural motif of collagen, and its integrity, which is known to affect cellular responses, is directly assessed in the decellularized ECM in our study. There are growing clinical interests in the use of decellularized biologic scaffolds for healing and regeneration of damaged tissues. The CHP technique introduced herein can help identify one of the key variables in the decellularization process — the molecular level destruction of collagen, and is expected to bring us one step closer to the understanding of how the decellularization process results in structural changes in the ECM and how that change influence the functional outcome of the ECM scaffolds. These new insights will ultimately lead to rationally optimized tissue decellularization protocols for specific regenerative applications.

 

Source:

http://doi.org/10.1016/j.actbio.2017.01.079

 

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