Development of Enzymatic-Resistant and Compliant Decellularized Extracellular Matrixes via Aliphatic Chain Modification for Bladder Tissue Engineering.

Here, we report the design and development of highly stretchable, compliant, and enzymatic-resistant transiently cross-linked decellularized extracellular matrixes (dECMs) (e.g., porcine small intestine submucosa/dSIS, urinary bladder matrix/dUBM, bovine pericardium/dBP, bovine dermis/dBD, and human dermis/dHD). Specifically, these dECMs were modified with long aliphatic chains (C9, C14, and C18). Upon modification, dECMs became significantly resistant to enzymatic degradation for extended periods, showed increased water contact angle (>20%-90%), and stretched >200% than their control counterparts. Modified dECMs are compliant, undergoing 100% elongation at only 0.3-0.5 MPa of applied tensile stress (∼10%-25% of their control counterparts), similar to the control bladder tissue. Furthermore, modified dECMs remain structurally stable at the physiological temperature with increased storage and loss modulus values but decreased tan δ values compared to their control counterparts. Although modification reduces cell adhesion, the gene expressions in polarized macrophages remain unchanged (e.g., TGFß, CD163, and CD86), except for the modified bovine pericardium (dBP) where a significant decrease in TNFα gene expression is observed. When implanted in the rat subcutaneous model, modified dECMs degraded relatively slowly and did not cause significant fibrotic tissue formation. The numbers of pro-regenerative macrophages increased to several folds in a later time point of evaluation. Modified dECM also supported the bladder wall regeneration with formations of the urothelium, lamina propria, blood vessels, and muscle bundles and reduced the occurrence of calculi formation by 50% in a rat bladder augmentation model. We anticipate that the enhanced stretchability, compliance, and physiological stability of dECMs indicate their suitability for urologic tissue regeneration.

Two-step sedimentation process for selection of microcapsules containing cell aggregates.

Microencapsulation technologies are being developed to protect transplanted islets from immune rejection, to reduce or even eliminate the need for immunosuppression. However, unencapsulated cells increase the chances of rejection and empty beads increase transplant volumes. Thus, separation processes were investigated to remove these byproducts based on density differences. The densities of islet-sized mouse insulinoma 6 (MIN6) cell aggregates and acellular 5% alginate beads generated via emulsification and internal gelation were ~ 1.065 and 1.042 g/ml, respectively. The separation of empty beads from those containing aggregates was performed by sedimentation under unit gravity in continuous gradients of polysucrose and sodium diatrizoate with density ranges of 1.032-1.045, 1.035-1.044, or 1.039-1.042 g/ml. The 1.039-1.042 g/ml gradient enabled recoveries of ~ 80% of the aggregate-containing beads while the other gradients recovered only ~ 60%. The bottom fraction of the 1.039-1.042 g/ml gradient contained beads with ~ 6% of their volume occupied by cell aggregates. Separation of unencapsulated aggregates from the aggregate-containing beads was then achieved by centrifugation of this purified fraction in a 1.055 g/ml density solution. Thus, these sedimentation-based approaches can effectively remove the byproducts of cell encapsulation.

The Specific Molecular Composition and Structural Arrangement of Eleutherodactylus Coqui Gular Skin Tissue Provide Its High Mechanical Compliance.

A male Eleutherodactylus Coqui (EC, a frog) expands and contracts its gular skin to a great extent during mating calls, displaying its extraordinarily compliant organ. There are striking similarities between frog gular skin and the human bladder as both organs expand and contract significantly. While the high extensibility of the urinary bladder is attributed to the unique helical ultrastructure of collagen type III, the mechanism behind the gular skin of EC is unknown. We therefore aim to understand the structure-property relationship of gular skin tissues of EC. Our findings demonstrate that the male EC gular tissue can elongate up to 400%, with an ultimate tensile strength (UTS) of 1.7 MPa. Species without vocal sacs, Xenopus Laevis (XL) and Xenopus Muelleri (XM), elongate only up to 80% and 350% with UTS~6.3 MPa and ~4.5 MPa, respectively. Transmission electron microscopy (TEM) and histological staining further show that EC tissues' collagen fibers exhibit a layer-by-layer arrangement with an uninterrupted, knot-free, and continuous structure. The collagen bundles alternate between a circular and longitudinal shape, suggesting an out-of-plane zig-zag structure, which likely provides the tissue with greater extensibility. In contrast, control species contain a nearly linear collagen structure interrupted by thicker muscle bundles and mucous glands. Meanwhile, in the rat bladder, the collagen is arranged in a helical structure. The bladder-like high extensibility of EC gular skin tissue arises despite it having eight-fold lesser elastin and five times more collagen than the rat bladder. To our knowledge, this is the first study to report the structural and molecular mechanisms behind the high compliance of EC gular skin. We believe that these findings can lead us to develop more compliant biomaterials for applications in regenerative medicine.