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Current strategies in urinary bladder augmentation include use of gastrointestinal segments, however, the technique is associated with inevitable complications. An acellular biologic scaffold seems to be a promising option for urinary bladder augmentation. The aim of this study was to evaluate the utility of bladder acellular matrix (BAM) for reconstruction of clinically significant large urinary bladder wall defects in a long-term porcine model. Urinary bladders were harvested from 10 pig donors. Biological scaffolds were prepared by chemically removing all cellular components from urinary bladder tissue. A total of 10 female pigs underwent hemicystectomy and subsequent bladder reconstruction with BAM. The follow-up study was 6 months. Reconstructed bladders were subjected to radiological, macroscopic, histological, immunohistochemical, and molecular evaluations. Six out of ten animals survived the 6-month follow-up period. Four pigs died during observation due to mechanical failure of the scaffold, anastomotic dehiscence between the scaffold and native bladder tissue, or occluded catheter. Tissue engineered bladder function was normal without any signs of postvoid residual urine in the bladder or upper urinary tracts. Macroscopically, graft shrinkage was observed. Urothelium completely covered the luminal surface of the graft. Smooth muscle regeneration was observed mainly in the peripheral graft region and gradually decreased toward the center of the graft. Expression of urothelial, smooth muscle, blood vessel, and nerve markers were lower in the reconstructed bladder wall compared to the native bladder. BAM seems to be a promising biomaterial for reconstruction of large urinary bladder wall defects. Further research on cell-seeded BAM to enhance urinary bladder regeneration is required., (© 2021 Wiley Periodicals LLC.)

Decellularization approaches have been commonly used as alternative techniques to reconstruct tissues. However, due to the complex tissue compartmentation of the larynx, the decellularization process may not retain the characteristics necessary for the successful recreation of the larynx. The aim of this study was to assess the effect of the decellularization process on the framework of the human cadaveric larynx generally and the cricoarytenoid joint specifically. In this work, five freshly frozen human cadaveric larynges were decellularized utilizing a protocol that was previously demonstrated to be effective in decellularizing a porcine larynx. The decellularization protocol included: biological, chemical, and physical decellularization methods. Each specimen served as its own control to assess changes after decellularization. Studies and measurements included: histological, using Hematoxylin and Eosin (H&E) and Live/Dead™ stains; DNA quantification; micro-computed tomography (μ-CT) imaging; and biomechanical testing of the cricoarytenoid joints. The decellularization protocol took 12 days for each specimen. Microscopy of H&E stained samples demonstrated substantial removal of cells with preservation of the extracellular matrix that was more evident in cartilage than muscle specimens. Confocal microscope images of Live/Dead™ stained specimens also demonstrated almost complete removal of cells. Pre-decellularization cartilage-DNA quantity range was 27.0 to 336.8 ng/mg while post-decellularization DNA quantity range was 0 to 30.4 ng/mg (p = 0.031). For muscles, pre-decellularization DNA quantity range was 150.0 to 3,384.6 ng/mg, while post-decellularization DNA quantity range was 0 to 45.5 ng/mg (p = 0.031). μ-CT demonstrated preservation of the cartilaginous framework with a slight reduction of cricoarytenoid joint space. Furthermore, μ-CT demonstrated no significant reduction in the Housefield unit (p = 0.25) and mineral density (p = 0.25) after decellularization. Biomechanical testing demonstrated a non-significant reduction of forces required for anterior displacement of the arytenoid (mean reduction of forces, 0.1 ± 0.2 N, p = 0.16) and forces required for posterior displacement of the arytenoid (mean reduction of forces, 0.2 ± 0.3 N, p = 0.05). This study demonstrates effective decellularization of human larynges as evidenced by significant DNA depletion and preservation of extracellular matrix, which are outcomes that are required for a biological scaffold to regenerate a non-immunogenic larynx. The decellularization process caused minimal weakness in the cricoarytenoid joints due to treatment with multiple detergents and enzymes in the decellularization protocol., (© 2021 Wiley Periodicals LLC.)

The idea of transplanting a sheet of laboratory-grown corneal endothelium dates back to 1978; however, the ideal scaffold is still lacking. We hypothesized that human crystalline lens capsules (LCs) could qualify as a scaffold and aimed to characterize the properties of this material for endothelial tissue engineering. LCs were isolated from donor eyes, stored at -80 °C, and decellularized with water and trypsin-EDTA. The decellularization was investigated by nuclear staining and counting and the capsule thickness was determined by optical coherence tomography and compared with Descemet's membrane (DM). Transparency was examined by spectrometry, and collagenase degradation was performed to evaluate its resistance to degradation. Cell-scaffold interaction was assessed by measuring focal adhesions surface area on LC and plastic. Finally, primary corneal endothelial cells were grown on LCs to validate the phenotype. Trypsin-EDTA decellularized most effectively, removing 99% of cells. The mean LC thickness was 35.76 ± 0.43 μm, whereas DM measured 25.93 ± 0.26 μm (p < .0001). Light transmission was 90% for both LC and DM. On a collagenase challenge, LC and amniotic membrane were digested after 13 hr, whereas DM was digested after 17 hr. The surface area of focal adhesions for cells grown on coated LCs was at least double that compared with other conditions, whereas tight junctions, ion pumps, and hexagonal morphology were well maintained when endothelial cells were cultured on LCs. In conclusion, LCs demonstrate excellent scaffolding properties for tissue engineering and sustain the cell phenotype and can be considered a suitable substrate for ocular tissue engineering or as a template for future scaffolds., (© 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.)

This study was performed to develop a method to decellularize human conjunctiva and to characterize the tissue in terms of its deoxyribose nucleic acid (DNA) content, tensile strength, collagen denaturation, basement membrane, extracellular matrix components and its potential to support conjunctival epithelial growth. Human conjunctival tissues were subjected to a decellularization process involving hypotonic detergent and nuclease buffers. Variations in sodium dodecyl sulfate concentration (0.05-0.5%, w/v) were tested to determine the appropriate concentration of detergent buffer. DNA quantification, collagen denaturation, cytotoxicity and tensile strength were investigated. Human conjunctival cell growth by explant culture on the decellularized tissue substrate was assessed after 28 days in culture. Samples were fixed and paraffin embedded for immunohistochemistry including conjunctival epithelial cell markers and extracellular matrix proteins. Conjunctival tissue from 20 eyes of 10 donors (age range 65-92 years) was used. Decellularization of human conjunctiva was achieved to 99% or greater DNA removal (p < 0.001) with absence of nuclear staining. This was reproducible at the lowest concentration of sodium dodecyl sulfate (0.05% w/v). No collagen denaturation (p = 0.74) and no difference in tensile strength parameters was demonstrated following decellularization. No significant difference was noted in the immunolocalization of collagen IV, laminin and fibronectin, or in the appearance of periodic acid-Schiff-stained basement membranes following decellularization. The decellularized tissue did not exhibit any cytotoxicity and explant culture resulted in the growth of stratified conjunctival epithelium. Allogeneic decellularized human conjunctiva can be successfully decellularized using the described protocol. It represents a novel substrate to support the expansion of conjunctival epithelium for ocular surface cellular replacement therapies. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

A combined physical-chemical protocol for whole full-thickness bladder decellularization is proposed, based on organ cyclic distention through repeated infusion/withdrawal of the decellularization agents through the urethra. The dynamic decellularization was intended to enhance cell removal efficiency, facilitating the delivery of detergents within the inner layers of the tissue and the removal of cell debris. The use of mild chemical detergents (hypotonic solution and non-ionic detergent) was employed to limit adverse effects upon matrix 3D ultrastructure. Inspection of the presence of residual DNA and RNA was carried out on decellularized matrices to verify effective cell removal. Histological investigation was focused on assessing the retention of adequate structural and functional components that regulate the biomechanical behaviour of the acellular tissue. Biomechanical properties were evaluated through uniaxial tensile loading tests of tissue strips and through ex vivo filling cystometry to evaluate the whole-organ mechanical response to a physiological-like loading state. According to our results, a dynamic decellularization protocol of 17 h duration with a 5 ml/min detergent infusion flow rate revealed higher DNA removal efficiency than standard static decellularization, resulting in residual DNA content < 50 ng/mg dry tissue weight. Furthermore, the collagen network and elastic fibres distribution were preserved in the acellular ECM, which exhibited suitable biomechanical properties in the perspective of its future use as an implant for bladder augmentation., (Copyright © 2013 John Wiley & Sons, Ltd.)