RESUMO
Pancreatic bioengineering is a potential therapeutic alternative for type 1 diabetes (T1D) in which the pancreas is decellularized, generating an acellular extracellular matrix (ECM) scaffold, which may be reconstituted by recellularization with several cell types to generate a bioartificial pancreas. No consensus for an ideal pancreatic decellularization protocol exists. Therefore, we aimed to determine the best-suited detergent by comparing sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), and Triton X-100 at different concentrations. Murine (n=12) and human pancreatic tissue from adult brain-dead donors (n=06) was harvested in accordance with Institutional Ethical Committee of the University of São Paulo Medical School (CEP-FMUSP) and decellularized under different detergent conditions. DNA content, histological analysis, and transmission and scanning electron microscopy were assessed. The most adequate condition for pancreatic decellularization was found to be 4% SDC, displaying: a) effective cell removal; b) maintenance of extracellular matrix architecture; c) proteoglycans, glycosaminoglycans (GAGs), and collagen fibers preservation. This protocol was extrapolated and successfully applied to human pancreas decellularization. The acellular ECM scaffold generated was recelullarized using human pancreatic islets primary clusters. 3D clusters were generated using 0.5×104 cells and then placed on top of acellular pancreatic slices (25 and 50 μm thickness). These clusters tended to connect to the acellular matrix, with visible cells located in the periphery of the clusters interacting with the ECM network of the bioscaffold slices and continued to produce insulin. This study provided evidence on how to improve and accelerate the pancreas decellularization process, while maintaining its architecture and extracellular structure, aiming at pancreatic bioengineering.
RESUMO
Protein aggregation plays important roles in life science as, for instance, those associated to neurodegenerative diseases. Although extensive efforts have been done to elucidate all the possible variables related to the aggregation process, much has yet to be done to unveil the main pathways governing protein assembling. In the current work, we induce bovine serum albumin (BSA) association, at pH 3.7, by adding sodium dodecyl sulfate (SDS) and sodium perfluorooctanoate (SPFO) surfactants to BSA solution as promoters of protein aggregation. Firstly, we combine molecular dynamic simulations (MD) to obtain a partially unfolded state of BSA's monomer at the acid pH and small angle X-ray scattering (SAXS) to validate the model. Interestingly, we found by SAXS that at pH 3.7 BSA monomers coexist with dimers in surfactant-free solution. Upon SDS and SPFO addition, the partial unfolded BSA may evolve to large aggregates depending on surfactant concentration. The threshold occurs at 30:1 and 45:1 SDS:BSA and SPFO:BSA molar ratio, respectively, according to turbidity, Thioflavin (ThT) fluorescence, synchrotron radiation circular dichroism (SRCD), SAXS and scanning electron microscopy (SEM) experiments. BSA aggregates are larger in the presence of SDS and structurally more defined upon SPFO binding. Isothermal titration calorimetry (ITC) results give support to infer that both surfactants initially bind to the BSA macromolecule forming a complex. Then, these complexes self-associate towards supramolecular aggregates. Taking into account the physicochemical characteristics of both surfactants and also MD simulations we may suggest that the higher rigidity of the fluorinated chains in respect to hydrogenated ones is crucial to induce more ordered and smaller BSA's aggregates. Our results thus evidence that the ligand structural flexibility might be of a key importance in the pathway of protein aggregation and may pave the way to better understand the early steps of neurodegenerative disorders.