The Quest for an Optimized Protocol for Whole-Heart Decellularization: A Comparison of Three Popular and a Novel Decellularization Technique and Their Diverse Effects on Crucial Extracellular Matrix Qualities

Abstract

Decellularized cardiac extracellular matrix (ECM) has been introduced as a template for cardiac tissue engineering, providing the advantages of a prevascularized scaffold that mimics native micro- and macroarchitecture to a degree difficult to achieve with synthetic materials. Nonetheless, the decellularization protocols used to create acellular myocardial scaffolds vary widely throughout the literature. In this study we performed a direct comparison of three previously described protocols while introducing and evaluating a novel, specifically developed fourth protocol, by decellularizing whole rat hearts through software-controlled automatic coronary perfusion. Although all protocols preserved the macroarchitecture of the hearts and all resulting scaffolds could successfully be reseeded with C2C12 myoblasts, assessing their biocompatibility for three-dimensional in vitro studies, we found striking differences concerning the microcomposition of the ECM scaffolds on a histological and biochemical level. While laminin could still be detected in all groups, other crucial ECM components, like elastin and collagen IV, were completely removed by at least one of the protocols. Further, only three protocols maintained a glycosaminoglycan content comparable to native tissue, whereas the remaining DNA content within the ECM varied highly throughout all four tested protocols. This study showed that the degree of acellularity and resulting ECM composition of decellularized myocardial scaffolds strongly differs depending on the decellularization protocol.

Customized Interface Biofunctionalization of Decellularized Extracellular Matrix: Toward Enhanced Endothelialization

“…Additionally, the hydrophobic aminohexanoic acid residues present in the synthetic peptides might also contribute to improve the retention of the peptide via hydrophobic interactions with the substrates. As previously reported, dECM scaffolds are rich in collagenous proteins, 1,2,8 which contain a high abundance of hydrophobic amino acids (i.e., alanine, glycine, and proline), and could therefore favor these interactions. According to the scale of hydrophilicity described by Hopp and Woods, 46 values of hydrophilicity for RGD, REDV, and YIGSR peptides are 6.3, 7.5, and -0.8, respectively, indicating a strong hydrophobic character for YIGSR.…”

“…N ative extracellular matrix (ECM) can be decellularized by standard detergent-based protocols under high preservation of its ultrastructural composition and microarchitecture 1 while eliminating cell-based antigens, such as a-Gal-epitopes or human leukocyte antigens, 2 which may trigger undesired immune response upon implantation to a host organism. 3 Therefore, decellularized ECM (dECM) is widely used as a platform for bioengineering of medical implants, having shown its suitability in a variety of preclinical as well as clinical models.…”

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

“…The current study verified that the protocol eliminated histologic staining for cell nuclei, showed that less than 50 ng of DNA was present per mg of dry weight of the tissue, and that any DNA was less than 200 bp in size 6 . Previously published methods for cardiac decellularization showed similar levels of decellularization in DNA staining and quantification 2,5,9,10 . Complete decellularization was accomplished in these studies using similar treatments of enzymes and detergents.…”

Coming to terms with tissue engineering and regenerative medicine in the lung

“…This is a major challenge. For example, several studies have demonstrated that ECM proteins such as collagen, laminin, elastin, and fibronectin can be retained following decellularization, whereas others have reported reduced levels of ECM proteins and important growth factors (1,27,152,162,217). Here, the protocol for decellularization appears to be important.…”