الفهرس 
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Abstract
Whole organ decellularization; a beacon of hope for a continuously growing population of end stage renal disease patients. The optimal whole kidney decellularization procedure yields a three-dimensional natural kidney extracellular matrix (ECM) scaffold, completely deprived from cellular materials that can elicit an immune response when implanted into a host. This decellularized ECM has the intrinsic spatial ability and biological cues that can promote cell adhesion, migration, proliferation and differentiation during recellularization. Patient’s own cells can be used for scaffold recellularization to create a functional immune-compatible kidney ready for transplantation.
Vascular perfusion of detergents as sodium dodecyl sulfate (SDS) is the most widely used whole kidney decellularization protocol. Unfortunately, both SDS and perfusion pressure have a significant detrimental effect on ECM ultrastructure, vasculature and matrisome proteins. Therefore, the optimal decellularization protocol must balance between complete ECM decellularization and preservation of ECM properties.
The aim of this study was to present a proper decellularization protocol for male rabbit kidney and to characterize the resultant decellularized ECM by evaluation of the structural integrity of its components as a natural scaffold ready for tissue engineering.
The study was conducted on ten male New Zealand White Rabbits (NZWR) of an average weight (1000- 1500 gm). Both kidneys were harvested and sorted into two groups:
• group I (Control group): included the ten right kidneys; processed immediately for examination and analysis.
• group II (Decellularization group): included the ten left kidneys; harvested carefully after cannulation and kept frozen until decellularization. At decellularization, kidneys were thawed, perfused with 0.5% sodium dodecyl sulfate (SDS) for 5-6 hours at room temperature until kidneys were completely white. These gross morphological changes were recorded and photographed. In addition, the weight of the kidneys was recorded and statistically analyzed at the start (fresh weight), after freezing/thawing and after complete decellularization. Finally, decellularized kidneys were processed for examination and analysis as those of control group.
Kidney specimens were processed for the following examinations:
• Histological examination of paraffin sections stained with: H&E, Feulgen nuclear reaction, modified McManus periodic acid Schiff’s technique (PAS), combined Alcian blue-PAS technique, modified Masson′s trichrome stain, Gordon & Sweets’ silver impregnation method and orcein stain.
• Immunohistochemical examination of paraffin sections stained with immunoperoxidase labeled streptavidin-biotin complex technique for detection of laminin, fibronectin, and collagen type IV.
• Examination of semithin sections stained with Toluidine blue stain.
• Examination of ultrathin sections with transmission electron microscope (TEM).
• Examination with scanning electron microscope (SEM).
Statistical analysis was performed for the following morphometric measurements:
• Mean area percentage of green collagen fibers in Masson’s trichrome stained sections.
• Mean area percentage of reticular fibers in Gordon and sweets silver impregnation-stained sections.
• Mean area percentage of internal elastic laminae of arcuate arteries in orcein stained sections.
• Mean optical density of glycoproteins (positive PAS reaction in PAS stained sections(.
• Mean optical density of glycosaminoglycans (GAGs) )positive Alcian blue reaction in Alcian blue-PAS stained sections(.
• Mean optical density of laminin positive reaction using anti-laminin antibody-stained sections.
• Mean optical density of fibronectin positive reaction using anti-fibronectin antibody-stained sections.
• Mean optical density of collagen IV positive reaction using anti-collagen IV antibody-stained sections.
• Mean thickness of Glomerular basement membranes measured during TEM examination of ultrathin sections.
Microscopic examination revealed complete decellularization of group II kidneys, confirmed by total absence of cellular materials and nuclear basophilia in H&E-stained sections and total absence of nuclear reaction in Feulgen nuclear reaction-stained sections. In addition, the histological profile of the decellularized kidneys’ architecture was apparently well preserved.
The integrity of different extracellular matrix components (collagen fibers, reticular fibers, elastic fibers, glycoproteins, glycosaminoglycans) was well preserved in both renal cortex and medulla of decellularization group kidneys. This was confirmed by statistical analysis of morphometric measurements recorded during examination of sections stained with modified Masson′s trichrome, Gordon & Sweets’ silver impregnation method, orcein stain, PAS and combined Alcian blue-PAS technique, respectively. The statistical analysis revealed no significant difference between ECM components of cortex and medulla of kidneys of decellularization group and those of control group.
In addition, the integrity of important ECM proteins (laminin, fibronectin, collagen IV) was apparently well preserved. Statistical analysis revealed no significant difference between the optical density of laminin and fibronectin in both renal cortex and medulla of both groups. The mean optical density of collagen IV also showed non-significant difference in renal cortex of both groups, although it was significantly decreased in renal medulla of decellularization group than that of renal medulla of control group.
The ultrastructural profile of kidneys of decellularization group was apparently well preserved during TEM examination. Also, TEM examination confirmed complete cell removal and continuity of glomerular basement membranes and tubular basement membranes. However, laminae rara externa and interna of glomerular basement membranes of decellularization group were not seen clearly as those of control group. Statistical analysis showed that thickness of glomerular basement membranes of decellularization group was significantly decreased when compared to that of control group.
In addition, SEM examination showed that the three-dimensional (3D) structure of glomeruli and tubules of decellularized kidneys was seen intact as that of control group.
Consequently, it was concluded that the presented protocol efficiently decellularized the male rabbit kidney while preserving the integrity of the resultant dECM.