Assessing the Effects of Bladder Decellularization Protocols on Extracellular Matrix (ECM) Structure, Mechanics, and Biology
Felix Yiu, B.S.1, Victoria Lee, B.S.1, Astha Sahoo, Undergrad1, Jonathan Shiba, Undergrad1, Nohemi Garcia-Soto, Undergrad1, George Aninwene II, Ph.D.1, Vijaya Pandey, Ph.D.2, James Wohlschlegel, Ph.D.2, Renea Sturm, M.D.1.
1Department of Urology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA, 2Proteome Research Center, University of California Los Angeles, Los Angeles, CA, USA.
Introduction: Acellular matrices are a key source of biologic scaffolds applied in tissue engineering with varied results. Decellularization methods and detergents vary widely, and there are few studies evaluating comparative effectiveness in maintaining nanotopography, mechanics, and protein content of these matrices in comparison to native healthy tissue. The aim of this study was to compare common bladder decellularization protocols, with a goal of defining the effects of each on cellular nuclei (goal ≥85% decrease), DNA removal (≥90%), maintenance of structure, preserved key extracellular matrix (ECM) proteins, and tensile properties.
Methods: Porcine bladders were decellularized with 0.5% Sodium Dodecyl Sulfate (SDS) or 0.25% Trypsin-hypotonic-Triton X-100 hypertonic (TT) protocols. Each bladder was distended at 4°C for 48 hours (h) with antibiotic solution, followed by agitation at 120rpm for 1) 48h at room temperature (RT) in SDS detergent or 2) trypsin (37°C, 2h), hypotonic (4°C, 22h), then hypertonic (RT, 24h) solutions for TT protocol. Finally, both were placed in DNase (RT, 3h) at 120rpm. Decellularization efficacy (DAPI, DNA quantification) and ECM structural maintenance (histology, scanning electron microscopy, total protein and protein assays) were assessed. Mechanical characterization was completed using an Instron 345C-1 mechanical tester (0.5 N, 10 mm/min). SDS vs TT data was directly compared using two-tailed unpaired t-tests. Native, TT, and SDS cohorts were compared using one-way ANOVA; Tukey’s post-hoc tests for among group differences. Data is presented as mean ± standard error.
Results: Effective nuclei removal was achieved by SDS and TT (Fig A, D). SDS more effectively maintained qualitative tissue architecture (B, C) as compared to TT. Target DNA removal was achieved with SDS but not TT (E). The tensile modulus increased, and elasticity decreased after decellularization; UTS was unaffected (F). BCA protein assay showed decreased total protein content by 83.8% ± 4.1 post-SDS and 65.2% ± 8.4 post-TT. Of note, remaining elastin content was 44.8% ± 0.8 post-SDS and 29.2% ± 0.2 post-TT of total elastin in native tissue normalized by sample weight (p<0.0001, native vs SDS and TT respectively).
Conclusion: The results indicated that SDS was superior to TT in achieving target decellularization efficacy and DNA removal, as well as maintaining tissue architecture. Although ultimate tissue strength and modulus was relatively preserved, elasticity was significantly decreased by both decellularization methods. Post-SDS protein extraction aligned with published content of bladder cellular and ECM protein [Marcal, 2012]. However, a decrease in elastin may contribute to the observed decrease in elasticity post-decellularization; a complete proteomic evaluation and additional assays including assessment of effects on collagen subtypes are ongoing. This study provides key comparative information to facilitate ECM evaluation in healthy versus diseased tissues and to inform the creation of bio-inspired scaffolds for tissue engineering.
Back to 2023 Abstracts