Abstract
Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM) must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS) was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic) preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic).
Highlights
Extracellular matrices (ECM) of decellularized tissues and/or organs have been widely used as biological biomaterials for tissue engineering purposes [1,2,3]
Both methods promoted cell removal (Figure 1C and D), the perfusion used in this study did not decrease the time required for tissue decellularization
Several decellularization protocols have been applied to different tissues and organs, such as kidney [11], liver [22], heart [23], lung [24], pancreas [25], and to human
Summary
Extracellular matrices (ECM) of decellularized tissues and/or organs have been widely used as biological biomaterials for tissue engineering purposes [1,2,3]. Decellularization protocols are tissue/organ specific and can be performed using chemical, physical and/or enzymatic methods [2,4]. An optimal decellularization protocol should guarantee complete cell and nucleic acids removal, preserve ECM physical structure and chemical composition [1,2] and cause no immune reactions in the host organism [5,6]. Decellularization processes inevitably affect ECM at different levels; distinct protocols must be tested and established for each tissue/organ of interest, where variables such as tissue thickness, ECM and cell density must be considered [5,7]. Decellularization has been tested in several tissues and organs such as murine hearts [9], murine and human lungs [10], porcine kidneys [11] and in different types of placentas [12,13]
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