Trypsin as enhancement in cyclical tracheal decellularization: Morphological and biophysical characterization.

There are different types of tracheal disorders (e.g. cancer, stenosis and fractures). These can cause respiratory failure and lead to death of patients. Several attempts have been made for trachea replacement in order to restore the airway, including anastomosis and implants made from synthetic or natural materials. Tracheal allotransplantation has shown high rejection rates, and decellularization has emerged as a possible solution. Decellularization involves the removal of antigens from cells in the organ or tissue, leaving a matrix that can be used as 3D cell-scaffold. Although this process has been used for tracheal replacement, it usually takes at least two months and time is critical for patients with tracheal disorders. Therefore, there is necessary to develop a tracheal replacement process, which is not only effective, but also quick to prepare. The aim of this research was to develop a faster trachea decellularization protocol using Trypsin enzyme and Ethylenediaminetetraacetic acid (EDTA) as decellularization agents. Three protocols of cyclic trachea decellularization (Protocols A, B, and C) were compared. Following Protocol A (previously described in the literature), 15 consecutive cycles were performed over 32 days. Protocol B (a variation of Protocol A) ­ EDTA being added ­ with 15 consecutive cycles performed over 60 days. Finally, Protocol C, with the addition of Trypsin as a decellularization agent, 5 consecutive cycles being performed over 10 days. For the three protocols, hematoxylin­eosin (H&E) staining and DNA residual content quantification were performed to establish the effectiveness of the decellularization process. Scanning Electron Microscopy (SEM) was used to observe the changes in porosity and microarrays. To evaluate the structural matrices integrity, Thermogravimetric Analysis (TGA) and biomechanical test were used. None of the protocols showed significant alteration or degradation in the components of the extracellular matrix (ECM). However, in Protocol C, more cellular components were removed in less time, making it the most efficient process. In addition, the cell tracking and viability was evaluated with chondrocytes seeding on the scaffold obtained by Protocol C, which showed an adequate cell scaffolding ability of this matrix.

Long-term follow-up of patients with type 1 diabetes transplanted with neonatal pig islets.

Pancreas transplantation is an option to achieve better metabolic control and decrease chronic complications in patients with diabetes. Xenotransplantation becomes an important alternative. In this study, we show the clinical outcome of patients with type 1 diabetes transplanted with neonatal pig islets without immunosuppression. In a longitudinal study of 23 patients with type 1 diabetes, who received porcine islets between 2000 and 2004, we registered demographic and clinical characteristics every 3 months and chronic complications evaluation yearly. Porcine C-peptide was measured in urine samples under basal conditions and after stimulation with l-arginine. More than 50% were female, median current age was 20·8 years, median diabetes duration at transplantation 5·5 years, median current diabetes duration 11 years and median time post-transplantation 5·7 years. Their media of glycosylated haemoglobin reduced significantly after the first transplantation. Insulin doses remain with a reduction greater than 33% in more than 50% of the patients. Before transplantation, 14 of the 21 patients presented mild chronic complications and currently only two patients presented these complications. Porcine C-peptide was present in all urine samples under basal conditions and increased post-stimulation with l-arginine. These patients achieved an excellent metabolic control after the first transplantation. This could explain, as well as the remaining function of transplanted cells, the low frequency of chronic complications compared to patients with similar diabetes duration and age.