Speaker
Description
Introduction: Urinary bladder regeneration using tissue engineering is one of the major challenges of reconstructive urology. Commonly used urinary bladder reconstruction techniques that utilize gastrointestinal tissues, are associated with many complications. One of the most promising alternative seems to be the use of bladder acellular matrix (BAM). The use of BAM in clinical practice requires the construction of an automated system that will enable bladder decellularization
in an efficient and reproducible manner.
The aim of this study was to develop an automated system that would enable a simple, reproducible and economically viable process for decellularization of the entire urinary bladder and to compare its effectiveness on pig and human urinary bladders.
Methodology: Porcine urinary bladders (n=20) were obtained from a local slaughterhouse and human bladders (n=10) were obtained from cadaveric donors. Firstly, we designed and constructed a homemade whole urinary bladder decellularization system on which a chemical decellularization protocol was developed on a porcine bladder(n=20) using trypsin-EDTA to remove urothelium, hypertonic buffer, Triton X-100 or SDS in hypertonic buffer to remove membranous and cytoplasmic materials. Then, on the basis of experience, a system for automatic decellularization was designed together with Zellwerk GmbH (Germany). Scaffold rinsing was optimised to remove cellular debris and of detergent residues. BAM acellularity was analyzed by histological stainings including HE and Trichrom. BAM structure was evaluated by Scanning Electron Microscopy. The presence of residual DNA was analysed using DAPI staining and molecular analysis. DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen, Germany) according to manufacturer’s protocol and quantified using NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, USA). In addition, BAM cytotoxicity against Adipose-Derived Mesenchymal Stem/Stromal Cells (AD-MSCs) in vitro was analyzed using MTT. A content of the BAM components, including collagen, elastin and laminin, were assessed using Collagen Assay Kit (Sigma-Aldrich, USA), Fastin™ Elastin (Biocolor Ltd., UK) and ELISA Kit for Laminin (Cloud-clone, USA). Additionally, the porcine BAM cytotoxicity, genotoxicity and systemic toxicity were analyzed according ISO-10993 standard in accredited laboratory.
Results: Decellularization was performed using an automated system on porcine and human bladders. According to the results, the automated decellularization system in which decellularization and washing were performed for 3 weeks showed the absence of cell nuclei, and high efficiency of DNA removal, a residual DNA content < 50ng/mg dry tissue weight for both bladders. The viability of AD-MSCs after incubation with BAMs extract obtained by this method was over 90% and 80% for porcine and human BAMs respectively. Additionally, tests in an accredited laboratory have shown that porcine BAM is non-cytotoxic, non-genotoxic and shows no systemic toxicity.
Conclusions: The research carried out allowed the design of an automated system for a simple, reproducible and cost-effective method of decellularizing the entire urinary bladder of both pigs and humans. Due to the wide variation in human bladder structure resulting from individual differences, decellularization of the human bladder is a more difficult process.
The present study was supported by the National Center for Research and Development (NCBR) in Poland under agreement no. LIDER/48//0195/L-9/17/NCBR/2018.
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