TABLE 4.
Summary table showing the approaches commonly adopted for evaluating the efficacy of decellularization processes and the methods of investigation for highlighting potentially dangerous tissue changes according to the future performance of the implant.
| Common approaches for the effectiveness evaluation of a decellularization process | ||
|---|---|---|
| Approach | Further details | |
| DNA | Commercial Kit for DNA extraction and quantification | n.d |
| Immunofluorescence assessment (qualitative) | Hoechst, Bisbenzimide H, Draq5, Live red dye, Picogreen | |
| Histological assessment (qualitative) | Feulgen stain | |
| Cells | Immunofluorescence assessment (qualitative) | DAPI (4′,6-diamidino-2-phenylindole) |
| Histological assessment (qualitative) | Ematoxylin and Eosin | |
| Xenoantigens | Immunohistochemistry assessment (qualitative) | anti-Neu5Gc, anti alpha-Gal and anti-SDa antibodies |
| ELISA test (Quantitative) | n.d | |
| Structural cell proteins | Immunohistochemistry assessment (qualitative) | anti-alpha-smooth muscle actin, beta-actin, vimentin, collagen VI and laminin antibodies |
| Hyaluronan fragments | Immunofluorescence assessment (qualitative) | Hyaluronan binding protein (HABP) probe biotin-conjugated |
| Collagen | Quantitative evaluation | Hydroxyproline-based assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), High-performance liquid chromatography with fluorescence detection (HPLC-FLD) |
| Histological assessment (qualitative) | Masson, Mallory and Van Gieson Trichromic staining, Picro Sirius Red | |
| Immunofluorescence assessment (qualitative) | anti-collagen I, II and III antibodies | |
| Elastin | Quantitative evaluation | Desmosine-base assay, NaOH extraction |
| Histological assessment (qualitative) | Verhoeff, Orcein, Weigert staining kit | |
| Immunofluorescence assessment (qualitative) | anti-elastin antibodies | |
| POST DECELLULARIZATION ASSESSMENT FOR TISSUES PERFORMANCES | ||
| Evaluation approach | Commonly considered parameters | |
| Mechanical properties | Bi- or uni-axial stress-strain test for patch-shape tissue | Ultimate Tensile Strength (UTS), Elongation, Young’s Modulus, Compressive Strength (CS), Suture retention Strength (SRS) |
| Pulse duplicator assessment for heart valve | Mean Pressure drop (MPD), Effective Orifice Area (EOA), Peak Pressure drop (PPD), Mean Regurgitation and Mean Energy Losses | |
| Thermal analysis | Thermogravimetric analysis (TGA) | Protein–water interactions and degradation temperatures |
| Differential scanning calorimetry (DSC) | Protein thermal transitions (glass transition and denaturation) | |
| Detergent residues | Quantitative chemical evaluation | Dimethylmethylene blue, reverse phase chromatography coupled with UV detection |