ABSTRACT
Developing patient-specific transplantable organs is a promising response to the increasing need of more effective therapies for patients with organ failure. Advances in tissue engineering strategies have demonstrated favorable results, including the use of decellularized hearts as scaffolds for cardiac engineering; however, there is a need to establish methods to characterize the cytotoxicity and blood compatibility of cardiac extracellular matrix (cECM) scaffolds created by decellularization. In this study, porcine hearts were decellularized in an automated perfusion apparatus utilizing sodium dodecyl sulfate (SDS) detergent. Residual SDS was measured by a colorimetric assay. Phosphate-buffered saline, distilled water (DW), and Triton X-100 washes were used to remove SDS. The efficiency of detergent removal was measured as a function of time. It was observed that using Triton-X 100 can nearly double the rate of SDS removal. An assay based on human blood hemolysis was developed to measure the remaining cytotoxicity of the cECM. The results from the hemolysis cytotoxicity assay were consistent with a standard live/dead assay using MS1 endothelial cells incubated with the cECM. This study demonstrated an effective, reliable, and relatively inexpensive method for determining the cytotoxicity and blood compatibility of decellularized cECM scaffolds.
Subject(s)
Hemolysis , Tissue Engineering/methods , Tissue Scaffolds , Animals , Collagen/analysis , Extracellular Matrix/physiology , Humans , Mice , Swine , Toxicity TestsABSTRACT
Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate repair or remodeling of numerous tissues, including the esophagus. The theoretically ideal scaffold for tissue repair is the ECM derived from the particular tissue to be treated, that is, site-specific or homologous ECM. The preference or potential advantage for the use of site-specific ECM remains unknown in the esophageal location. The objective of the present study was to characterize the in vitro cellular response and in vivo host response to a homologous esophageal ECM (eECM) versus nonhomologous ECMs derived from small intestinal submucosa and urinary bladder. The in vitro response of esophageal stem cells was characterized by migration, proliferation, and three-dimensional (3D) organoid formation assays. The in vivo remodeling response was evaluated in a rat model of esophageal mucosal resection. Results of the study showed that the eECM retains favorable tissue-specific characteristics that enhance the migration of esophageal stem cells and supports the formation of 3D organoids to a greater extent than heterologous ECMs. Implantation of eECM facilitates the remodeling of esophageal mucosa following mucosal resection, but no distinct advantage versus heterologous ECM could be identified.
Subject(s)
Esophagus/physiology , Extracellular Matrix/metabolism , Organ Specificity , Animals , Cell Proliferation/drug effects , Chemotaxis/drug effects , Esophagus/drug effects , Esophagus/surgery , Female , Hydrogels/pharmacology , Keratins/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Mucous Membrane/physiology , Organoids/cytology , Organoids/drug effects , Rats, Sprague-Dawley , Stem Cells/cytology , Stem Cells/drug effects , Sus scrofa , Tissue Scaffolds/chemistryABSTRACT
Whole heart decellularization combined with patient-specific cells may prove to be an extremely valuable approach to engineer new hearts. Mild detergents are commonly used in the decellularization process, but are known to denature and solubilize key proteins and growth factors and can therefore be destructive to the extracellular matrix (ECM) during the decellularization process. In this study, the decellularization of porcine hearts was accomplished in 24 h with only 6 h of sodium dodecyl sulfate exposure and 98% DNA removal. Automatically controlling the pressure during decellularization reduced the detergent exposure time while still completely removing immunogenic cell debris. Stimulation of macrophages was greatly reduced when comparing native tissue samples to the processed ECM. Complete cell removal was confirmed by analysis of DNA content. General collagen and elastin preservation was demonstrated. Glycosaminoglycans and collagen quantification both showed no significant differences in content after decellularization. The compression elastic modulus of the ECM after decellularization was lower than native at low strains, but there was no significant difference at high strains. Polyurethane casts of the vasculature of native and decellularized hearts demonstrated that the microvasculature network was preserved after decellularization. A static blood thrombosis assay using bovine blood was also developed. Finally, the recellularization potential of the ECM samples was demonstrated by reseeding cardiac fibroblasts and endothelial cells on the myocardium and endocardium samples.
