Assessing the effects of bladder decellularization protocols on extracellular matrix (ECM) structure, mechanics, and biology

Abstract

Introduction

Acellular matrices have historically been applied as biologic scaffolds in surgery, wound care, and tissue engineering, albeit with inconsistent outcomes. One aspect that varies widely between products is the selection of decellularization protocol, yet few studies assess comparative effectiveness of these protocols in preserving mechanics, and protein content. This study characterizes bladder acellular matrix (BAM) using two different detergent and enzymatic protocols, evaluating effects on nuclei and DNA removal (≥90%), structure, tensile properties, and maintenance of extracellular matrix proteins.

Methods

Porcine bladders were decellularized with 0.5% Sodium Dodecyl Sulfate (SDS) or 0.25% Trypsin-hypotonic-Triton X-100 hypertonic (TT)-based agitation protocols, followed by DNase/RNase agents. Characterization of BAM included decellularization efficacy (DAPI, DNA quantification), structure (histology and scanning electron microscopy), tensile testing (Instron 345C-1 mechanical tester), and protein presence and diversity (mass spectrometry). SDS and TT data was directly compared to the same native bladder using two-tailed paired t-tests. Native, TT, and SDS cohorts for tensile testing were compared using one-way ANOVA; Tukey's post-hoc tests for among group differences.

Results

Effective nuclei removal was achieved by SDS- and TT-based protocols. However, target DNA removal was achieved with SDS but not TT. SDS more effectively maintained qualitative tissue architecture compared to TT. The tensile modulus of the TT cohort increased, and stretchability decreased after decellularization in both SDS and TT. UTS was unaffected by either protocol. Higher overall diversity and quantity of core matrisome and matrisome-associated proteins was maintained in the SDS vs TT cohort post-decellularization.

Conclusion

The results indicated that detergent selection affects multiple aspects of the resultant BAM biologic product. In the selected protocols, SDS was superior to TT efficacy, and maintenance of gross tissue architecture as well as maintenance of ECM proteins. Decellularization increased scaffold resistance to deformation in both cohorts. Future studies applying biologic scaffolds must consider the processing method and agents used to ensure that materials selected are optimized for characteristics that will facilitate effective translational use.

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