Introduction and Objective: A variety of congenital and acquired pathologies resulting in loss of bladder capacity or function require surgical repair (1). The extracellular matrix (ECM) of decellularized animal tissues represents a key tissue engineering approach that provides a biologic scaffold material for the repair and reconstruction of damaged tissues or organs (2). Acellular bladder matrix was first developed in order to circumvent the complications that arose with utilizing vascularized bowel segments for bladder reconstruction – namely the incompatibility of bowel tissue with long term exposure to urine (3). A variety of protocols for decellularizing bladder tissues have been previously described (4-6), but the literature continues to lack a comparison and elucidation of the effects of different methods of decellularization on the microenvironments inherent to the tissue. The aim of this study was to compare and contrast decellularization protocols to select for one that would achieve target cellular product and DNA removal to prevent immunogenicity while maintaining ECM structure, mechanics, and protein content. We aimed to achieve a minimum of 90% DNA removal as well as removal of cellular proteins (representing an estimated 86% of total protein). A profile of porcine urinary bladder protein has shown that approximately 14% consists of ECM protein, which we aim to preserve (7). As ECM integrity directly impacts the mechanical properties of a tissue (8), we aimed to achieve an ultimate tensile strength after decellularization that is not statistically different from native tissue.The preservation of structural, mechanical, and biological cues in the scaffold are critical to inform development of an ideal substrate for tissue-specific regeneration.

Methods: Intact porcine bladders from adult healthy males obtained from an abbatoir were either analyzed as native tissue, or decellularized with a 2 day protocol utilizing either 0.5% Sodium Dodecyl Sulfate (SDS) (9) or 0.25% Trypsin followed by hypotonic solution and hypertonic solution with Triton X-100. For either method, bladders were subsequently incubated in DNase/RNase (room temperature, 3 hours) (10). Decellularization efficacy (DAPI, DNA content analysis) and ECM structural maintenance (histology, scanning electron microscopy (SEM) and total protein) were assessed. Mechanical characterization was completed (Instron 345C-1). Tissue decellularized with Sodium Dodecyl Sulfate (SDS) was compared to tissue decellularized with the Trypsin/Triton X-100 protocol (TT) using qualitative measures and two-tailed T-tests. Data is presented as mean (± standard error of the mean). Alternatively, when appropriate, Native, TT and SDS tissues were compared using one-way ANOVA, with differences among groups determined with Tukey’s post-hoc test.

Results: In both SDS and TT decellularized bladders, H&E showed the absence of nuclei. SEM and H&E images demonstrated that only SDS treated tissue maintained both surface integrity and the architecture of the mucosa, submucosa and muscularis propria. Quantification of DNA content (ng/ul) per mg of tissue showed an average of 51.6% (± 9.0) DNA removal with the Trypsin protocol and 90.0% (± 4.5) DNA removal with the SDS protocol (p=0.02). A one way ANOVA revealed a statistically significant difference in mean nuclei counts between experimental groups (F(2,11)=30.71, p<0.0001). Tukey’s post-hoc tests revealed a statistically significant decrease in nuclei count between Native and TT, and Native and SDS, but no significant difference between TT and SDS. BCA protein assay demonstrated an average decrease of 83.8% (± 4.1) in total protein content with the SDS protocol while the Trypsin protocol resulted in a 65.29% (±8.4) decrease (p=0.09). The tensile testing data indicated that although elastic modulus increased after decellularization, the overall bladder tensile strength at failure was not significantly changed for either decellularization method.

Conclusions: The results indicated that SDS was superior to TT in decellularization efficacy and achieved target parameters for DNA removal (minimum 90%). While percentage elongation did decrease after decellularization, ultimate tensile strength of the tissue did not change significantly with decellularization by either method, suggesting that the ECM integrity of the tissues were maintained. Qualitative analysis of architectural elements of the bladder tissue indicated that SDS better preserved ultrastructure as compared to TT. The SDS protocol removed approximately 84% of total protein, which aligns with the removal of cellular protein. Although SDS removes more total protein content from the tissue, further investigation is required in the form of proteomics to elucidate the ECM components in native and decellularized tissue to ensure that essential components are maintained.

References:

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