Decellularized tissue is an important source for biological tissue engineering. Evaluation of the quality of decellularized tissue is performed using scanned images of hematoxylin-eosin stained (H&E) tissue sections and is usually dependent on the observer. The first step in creating a tool for the assessment of the quality of the liver scaffold without observer bias is the automatic segmentation of the whole slide image into three classes: the background, intralobular area, and extralobular area. Such segmentation enables to perform the texture analysis in the intralobular area of the liver scaffold, which is crucial part in the recellularization procedure. Existing semi-automatic methods for general segmentation (i.e., thresholding, watershed, etc.) do not meet the quality requirements. Moreover, there are no methods available to solve this task automatically. Given the low amount of training data, we proposed a two-stage method. The first stage is based on classification of simple hand-crafted descriptors of the pixels and their neighborhoods. This method is trained on partially annotated data. Its outputs are used for training of the second-stage approach, which is based on a convolutional neural network (CNN). Our architecture inspired by U-Net reaches very promising results, despite a very low amount of the training data. We provide qualitative and quantitative data for both stages. With the best training setup, we reach 90.70% recognition accuracy.

In this retrospective study on 67 patients with hepatocellular carcinoma (HCC), after tumor resection, we evaluated the significance of CD3+ and CD8+ T-lymphocytes and CD20+ B-lymphocytes in tumor and non-tumor liver for time to recurrence (TTR), disease-free survival (DFS) and overall survival. After immunohistochemical staining, the density of nucleated lymphocyte profiles (Q_A) was estimated stereologically in the tumor center (TC), inner margin (inn M), outer margin (out M), peritumor and non-tumor liver. In TC, intermediate and high Q_A of CD8+ cells predicted longer TTR, whereas CD3+ and CD20+ were predictive only at high Q_A. DFS was predicted by high Q_A of CD3+, CD8+ and CD20+ cells in TC. The inn M harbored smaller Q_A of CD3+, CD8+ and CD20+ lymphocytes than out M. In contrast to out M, high T-cells’ Q_A and intermediate and high B-cell Q_A in inn M predicted longer TTR and DFS. High inn M/out M Q_A ratios of CD3+ and CD20+ cells were associated with longer TTR and DFS, whereas high inn M/out M Q_A ratio of CD8+ was predictive only for DFS. Patients with intermediate-high Q_A of combined CD8+ and CD20+ cells in inn M showed longer TTR and DFS, compared to CD8+-high or CD20+-high alone. Our findings highlight overall heterogeneity of the tumor invasive margin, the importance of inn M, and the predictive role of B-cells.

Anastomotic leakage is a dreadful complication in colorectal surgery. It has a negative impact on postoperative mortality, long term life quality and oncological results. Nanofibrous polycaprolactone materials have shown pro-healing properties in various applications before. Our team developed several versions of these for healing support of colorectal anastomoses with promising results in previous years. In this study, we developed highly porous biocompatible polycaprolactone nanofibrous patches. We constructed a defective anastomosis on the large intestine of 16 pigs, covered the anastomoses with the patch in 8 animals (Experimental group) and left the rest uncovered (Control group). After 21 days of observation we evaluated postoperative changes, signs of leakage and other complications. The samples were assessed histologically according to standardized protocols. The material was easy to work with. All animals survived with no major complication. There were no differences in intestinal wall integrity between the groups and there were no signs of anastomotic leakage in any animal. The levels of collagen were significantly higher in the Experimental group, which we consider to be an indirect sign of higher mechanical strength. The material shall be further perfected in the future and possibly combined with active molecules to specifically influence the healing process.

The present experiments were performed to evaluate if increased heart tissue concentration of fatty acids, specifically myristic, palmitic and palmitoleic acids that are believed to promote physiological heart growth, can attenuate the progression of unloading-induced cardiac atrophy in rats with healthy and failing hearts. Heterotopic abdominal heart transplantation (HTx) was used as a model for heart unloading. Cardiac atrophy was assessed from the ratio of the native- to-transplanted heart weight (HW). The degree of cardiac atrophy after HTx was determined on days 7, 14, 21 and 28 after HTx in recipients of either healthy or failing hearts. HTx of healthy hearts resulted in 23±3, 46±3, 48±4 and 46±4 % HW loss at the four time-points. HTx of the failing heart resulted in even greater HW losses, of 46±4, 58±3, 66±2 and 68±4 %, respectively (P<0.05). Activation of “fetal gene cardiac program” (e.g. beta myosin heavy chain gene expression) and “genes reflecting cardiac remodeling” (e.g. atrial natriuretic peptide gene expression) after HTx was greater in failing than in healthy hearts (P<0.05 each time). Exposure to isocaloric high sugar diet caused significant increases in fatty acid concentrations in healthy and in failing hearts. However, these increases were not associated with any change in the course of cardiac atrophy, similarly in healthy and post-HTx failing hearts. We conclude that increasing heart tissue concentrations of the fatty acids allegedly involved in heart growth does not attenuate the unloading-induced cardiac atrophy.

Tissue decellularization has emerged as a technique to provide an acellular, non-immunogenic scaffold that preserves the morphological features of native tissue. To study the possible effects of decellularization, investigating the mechanical behavior and the protein composition is crucial. In this study, we performed extension-inflation tests on native and decellularized porcine vena cava and investigated their microstructure using multiphoton microscopy. The mechanical behavior of both groups showed typical pressure-stretch curves of vascular structures with viscoelastic and nonlinear features. Importantly, no significant differences were found at inflation of 10, 20 and 30 mmHg, although some variability was observed in the decellularized scaffolds. When analyzing the results of the vessel wall multiphoton microscopy investigations, it was found that collagen fibers were packed in tortuous bundles in the media, but scattered in the adventitia. The fibers were oriented around 72° from the circumferential direction for both groups and at the same time equally distributed out-of-plane. Moreover, the collagen fibers diameter for media and adventitia was around 4 µm. Tortuosity and straightness were the same in the adventitia; however, the situation was different in the media, where the fibers in native samples were straighter than in decellularized scaffolds. Our findings show the potential of our protocol to obtain venous scaffolds that could be used for vascular reconstruction, as their mechanical properties are largely comparable to those of their native counterparts. The detailed analysis of the microstructure also represents a first step towards better understanding the physiology of the vessels and replicating these conditions in silico. STATEMENT OF SIGNIFICANCE: Tissue engineering provides a scaffold as substrate for in vitro cells seeding. Decellularization completely removes immunogenic cellular components, preserving the organ ultrastructure. Consequently, decellularized scaffolds provide a natural microenvironment for cell repopulation and facilitate functional recovery in vitro. We have comprehensively characterized the decellularized porcine vena cava by comparing its mechanical properties and microstructural characteristics with its native counterpart. Extension-inflation testing is considered a method to mimic stresses and stretches in vivo. Since no significant differences were found between native and decellularized tissue, these scaffolds show some potential. Moreover, this study was expanded to include microstructural characterization of collagen fibers using multi-photon microscopy, making it the first of its kind dedicated to biomechanical and microstructural evaluation of decellularized veins.