Structural and mechanical analysis of decellularizing rabbit kidney using SDS and SDS-Triton as detergents

Structural and mechanical analysis of decellularizing rabbit kidney using SDS and SDS-Triton as detergents
Yashar Rezaiepour ,^1,* Abdolmohammad Kajbafzadeh,²
1. Tarbiat modares university
2. Tehran University of Medical Sciences
Introduction: Tissue engineering is a multidisciplinary field that uses engineering, materials, and cells in restoring tissue functionality. Scaffolds play the role of ECM so they should have the biological and structural features of the original tissue. Recently the use of decellularized tissues as a natural scaffold has been studied. Decellularization is the process of isolating cells from an ECM that leads to a scaffold. Acellular ECM is the most mimetic scaffold to the natural ECM in engineering different tissues, including ligament, tendon, etc. Various methods have been proposed for tissue decellularization. Triton X100 and SDS are the most common nonionic and ionic detergents. Tissue differences need optimization of the decellularization method according to the tissue in a manner that does not harm the main components of the ECM. Recently, some researches aimed to investigate the effects of different decellularization protocols on ECM structure and composition. In this paper, rabbit kidneys have been decellularized by SDS and SDS-Triton perfusion. Then the two protocols compared by using fluorescence staining, calculating the remaining DNA, and determining mechanical properties
Methods: Decellularization Rabbit kidneys were connected to a peristaltic pump by cannulation through the renal artery. The first group perfused using SDS 2% for 96hr. The other group first perfused with SDS for 94hr and then perfused with Triton X100 for further 2hr. Optical microscopy samples, sliced with 3mm thickness (for formalin diffusion) and fixed in 48hr. Then the samples dehydrated using a series of increasing alcohol concentration paraffinized and sliced into extremely thin slices using a microtome. H&E and DAPI staining are used for histology and labeling DNA and evaluating decellularization process respectively. Glutaraldehyde (Merck) is used for fixing tissues for electron microscopy. Then samples dehydrated, dried using freeze drier for 24h and coated with gold to be conductive. Compression testing with a 50% deformation was used for determining the mechanical properties of the decellularized tissue.
Results: SEM images show the scaffold structure. The images show that cell removal leads to pores with a size of around 75 microns. Also, it is seen that Different parts of the kidney have their characteristic topography. DNA staining reveals that using SDS and Triton simultaneously as a decellularization agent leads to less DNA content in comparison to SDS. H&E staining figures of the normal kidney are representative of proximal and distal tubules, bowman’s capsule and glomerulus. Also, it can be seen that in both of the protocols the structure of the basic components of the kidney is well preserved in comparison to the normal kidney. Triton-SDS processed scaffolds have the least DAPI staining which shows the maximum cell and debris removal.
Conclusion: H&E staining shows that SDS and SDS-Triton agents do not change ECM structure seriously and the structure is the same as normal tissue. But comparing DAPI staining reveals that Triton helps to more material extraction from the ECM. Comparing the Compressive strength of the normal and acellular tissue shows that since the cells are harder than the ECM, cell removal causes strength loss. Also, using SDS – Triton resulted in a weaker scaffold than the ECM decellularized using Triton. Based on the above-mentioned results Triton-SDS is considered the most effective strategy for cell removal and structure preservation
Keywords: Natural scaffold- acellular matrix- Tissue engineering - regenerative medicine