Anatomical biliary reconstruction as an ultimum refugium for selective cases-History and current state of knowledge.

Reconstruction of extrahepatic bile ducts is a staple procedure of HPB surgery. The current standard for most cases is a nonanatomical bilioenteric reconstruction, a satisfactory option for the majority of patients. However, it cannot be used for a small number of selective cases (short bowel syndrome, severe abdominal adhesions), where an anatomical reconstruction with or without an interponate can be used. This review summarizes current knowledge about tissue and material usage for experimental and clinical anatomical bile duct reconstruction in the last 100 years. A Pubmed database was searched for published articles about anatomical extrahepatic bile duct reconstruction in experimental and clinical settings ranging from 1920 to 2022. To date, the truly optimal interponate material has not yet been found. However, evidence reveals important properties of such material, most importantly its biodegradability and neovascularization in the recipient's body. The role of internal bile duct stenting for anatomical reconstruction seems important for the outcome. Anatomical reconstruction of extrahepatic bile ducts is an uncommon but usable technique in unique cases when a nonanatomical reconstruction cannot be done. The optimal properties of interponate material for anatomical bile duct reconstruction have been more clarified, although further research is required.

Cross-species variability in lobular geometry and cytochrome P450 hepatic zonation: insights into CYP1A2, CYP2D6, CYP2E1 and CYP3A4.

There is a lack of systematic research exploring cross-species variation in liver lobular geometry and zonation patterns of critical drug-metabolizing enzymes, a knowledge gap essential for translational studies. This study investigated the critical interplay between lobular geometry and key cytochrome P450 (CYP) zonation in four species: mouse, rat, pig, and human. We developed an automated pipeline based on whole slide images (WSI) of hematoxylin-eosin-stained liver sections and immunohistochemistry. This pipeline allows accurate quantification of both lobular geometry and zonation patterns of essential CYP proteins. Our analysis of CYP zonal expression shows that all CYP enzymes (besides CYP2D6 with panlobular expression) were observed in the pericentral region in all species, but with distinct differences. Comparison of normalized gradient intensity shows a high similarity between mice and humans, followed by rats. Specifically, CYP1A2 was expressed throughout the pericentral region in mice and humans, whereas it was restricted to a narrow pericentral rim in rats and showed a panlobular pattern in pigs. Similarly, CYP3A4 is present in the pericentral region, but its extent varies considerably in rats and appears panlobular in pigs. CYP2D6 zonal expression consistently shows a panlobular pattern in all species, although the intensity varies. CYP2E1 zonal expression covered the entire pericentral region with extension into the midzone in all four species, suggesting its potential for further cross-species analysis. Analysis of lobular geometry revealed an increase in lobular size with increasing species size, whereas lobular compactness was similar. Based on our results, zonated CYP expression in mice is most similar to humans. Therefore, mice appear to be the most appropriate species for drug metabolism studies unless larger species are required for other purposes, e.g., surgical reasons. CYP selection should be based on species, with CYP2E1 and CYP2D6 being the most preferable to compare four species. CYP1A2 could be considered as an additional CYP for rodent versus human comparisons, and CYP3A4 for mouse/human comparisons. In conclusion, our image analysis pipeline together with suggestions for species and CYP selection can serve to improve future cross-species and translational drug metabolism studies.

Protein expression and localization of ABC transporters in pancreatic adenocarcinoma: Prognostic role of ABCC8.

BACKGROUND: ATP-binding cassette (ABC) transporters translocate various substances across cellular membranes. Their deregulation may cause cancer drug resistance or perturbations in the supply of building blocks for cancer cells and modify patients' prognosis. This study investigated protein expression and cellular localization of the previously suggested putative prognostic biomarkers - ABCB2/TAP1, ABCC7/CFTR, ABCC8/SUR1, and ABCD4 in patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: Protein expression and localization were assessed by immunohistochemistry in formalin-fixed paraffin-embedded primary tumor tissue blocks of 61 PDAC patients and associated with clinical data and the survival of patients. RESULTS: No CFTR protein expression was observed in PDAC, while TAP1 and ABCC8 were expressed predominantly in the cytoplasm of tumor cells. Most samples (81 %) had detectable both membranous and cytoplasmic ABCD4 staining and 42 % had ABCD4 expressed in the apical orientation. Negative membranous ABCD4 staining was significantly more frequent in advanced stage III or IV tumors (p = 0.022). Small or medium counts of individual ABCC8-positive cells in the stroma surrounding tumor tubules were also more often found in stage III or IV (p = 0.044). Patients with moderate or strong ABCC8 cytoplasmic staining intensity in tumor cells had a 3.5-fold higher risk of disease progression than those with weak staining (p = 0.002). CONCLUSIONS: The study shows for the first time that the cytoplasmic ABCC8 protein expression has prognostic value in PDAC.

Decellularization of Porcine Carotid Arteries: From the Vessel to the High-Quality Scaffold in Five Hours.

The use of biologically derived vessels as small-diameter vascular grafts in vascular diseases is currently intensely studied. Vessel decellularization provides a biocompatible scaffold with very low immunogenicity that avoids immunosuppression after transplantation. Good scaffold preservation is important as it facilitates successful cell repopulation. In addition, mechanical characteristics have to be carefully evaluated when the graft is intended to be used as an artery due to the high pressures the vessel is subjected to. Here, we present a new and fast decellularization protocol for porcine carotid arteries, followed by investigation of the quality of obtained vessel scaffolds in terms of maintenance of important extracellular matrix components, mechanical resistance, and compatibility with human endothelial cells. Our results evidence that our decellularization protocol minimally alters both the presence of scaffold proteins and their mechanical behavior and human endothelial cells could adhere to the scaffold in vitro. We conclude that if a suitable protocol is used, a high-quality decellularized arterial scaffold of non-human origin can be promptly obtained, having a great potential to be recellularized and used as an arterial graft in transplantation medicine.

Using virtual microscopy for the development of sampling strategies in quantitative histology and design-based stereology.

Only a fraction of specimens under study are usually selected for quantification in histology. Multilevel sampling or tissue probes, slides and fields of view (FOVs) in the regions of interest (ROIs) are required. In general, all parts of the organs under study should be given the same probability to be taken into account; that is, the sampling should be unbiased on all levels. The objective of our study was to provide an overview of the use of virtual microscopy in the context of developing sampling strategies of FOVs for stereological quantification. We elaborated this idea on 18 examples from multiple fields of histology, including quantification of extracellular matrix and muscle tissue, quantification of organ and tumour microvessels and tumour-infiltrating lymphocytes, assessing osseointegration of bone implants, healing of intestine anastomoses and osteochondral defects, counting brain neurons, counting nuclei in vitro cell cultures and others. We provided practical implications for the most common situations, such as exhaustive sampling of ROIs, sampling ROIs of different sizes, sampling the same ROIs for multiple histological methods, sampling more ROIs with variable intensities or using various objectives, multistage sampling and virtual sampling. Recommendations were provided for pilot studies on systematic uniform random sampling of FOVs as a part of optimizing the efficiency of histological quantification to prevent over- or undersampling. We critically discussed the pros and cons of using virtual sections for sampling FOVs from whole scanned sections. Our review demonstrated that whole slide scans of histological sections facilitate the design of sampling strategies for quantitative histology.

Decellularized xenogeneic scaffolds in transplantation and tissue engineering: Immunogenicity versus positive cell stimulation.

Seriously compromised function of some organs can only be restored by transplantation. Due to the shortage of human donors, the need to find another source of organs is of primary importance. Decellularized scaffolds of non-human origin are being studied as highly potential biomaterials for tissue engineering. Their biological nature and thus the ability to provide a naturally-derived environment for human cells to adhere and grow highlights their great advantage in comparison to synthetic scaffolds. Nevertheless, since every biomaterial implanted in the body generates immune reaction, studying the interaction of the scaffold with the surrounding tissues is necessary. This review aims to summarize current knowledge on the immunogenicity of semi-xenografts involved in transplantation. Moreover, positive aspects of the interaction between xenogeneic scaffold and human cells are discussed, focusing on specific roles of proteins associated with extracellular matrix in cell adhesion and signalling.

Double-layered Nanofibrous Patch for Prevention of Anastomotic Leakage and Peritoneal Adhesions, Experimental Study.

BACKGROUND/AIM: Anastomotic leakage is a feared complication in colorectal surgery. Postoperative peritoneal adhesions can also cause life-threatening conditions. Nanofibrous materials showed their pro-healing properties in various studies. The aim of the study was to evaluate the impact of double-layered nanofibrous materials on anastomotic healing and peritoneal adhesions formation. MATERIALS AND METHODS: Two versions of double-layered materials from polycaprolactone and polyvinyl alcohol were applied on defective anastomosis on the small intestine of healthy pigs. The control group remained with uncovered defect. Tissue specimens were subjected to histological analysis and adhesion scoring after 3 weeks of observation. RESULTS: The wound healing was inferior in the experimental groups, however, no anastomotic leakage was observed and the applied material always kept covering the defect. The extent of adhesions was larger in the experimental groups. CONCLUSION: Nanofibrous materials may prevent anastomotic leakage but delay healing.

Influence of Mesenchymal Stem Cell Administration on The Outcome of Partial Liver Resection in a Porcine Model of Sinusoidal Obstruction Syndrome.

BACKGROUND: In patients with colorectal liver metastases, the possibility for radical liver resection can be limited by oxaliplatin-induced sinusoidal obstruction syndrome (SOS). This study investigates the potential of mesenchymal stem cells (MSC) to improve the outcome of liver resections in pigs with SOS. MATERIALS AND METHODS: SOS was induced in all animals (n=20) on day 0. Animals in the experimental group (n=8) received allogeneic MSC on day 7. Liver resection was performed in all animals on day 14 and the animals were observed until day 28. Ultrasound volumetry, biochemical analysis and histological examination of liver parenchyma was performed during the follow-up period. RESULTS: Six animals from the control group died prematurely, while all animals survived in the experimental group. According to histology, biochemical analysis and ultrasound volumetry, there were no significant differences between the groups documenting the effect of MSC. CONCLUSION: Single dose allogeneic MSC administration improved survival of animals with SOS undergoing partial liver resection. Further experiments with different timing of liver resection and MSC administration should be performed to investigate the effect of MSC in more detail.

Semantic Segmentation of Intralobular and Extralobular Tissue from Liver Scaffold H&E Images.

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.

Novel morphological multi-scale evaluation system for quality assessment of decellularized liver scaffolds.

Decellularized scaffolds can serve as an excellent three-dimensional environment for cell repopulation. They maintain tissue-specific microarchitecture of extracellular matrix proteins with important spatial cues for cell adhesion, migration, growth, and differentiation. However, criteria for quality assessment of the three-dimensional structure of decellularized scaffolds are rather fragmented, usually study-specific, and mostly semi-quantitative. Thus, we aimed to develop a robust structural assessment system for decellularized porcine liver scaffolds. Five scaffolds of different quality were used to establish the new evaluation system. We combined conventional semi-quantitative scoring criteria with a quantitative scaffold evaluation based on automated image analysis. For the quantitation, we developed a specific open source software tool (ScaffAn) applying algorithms designed for texture analysis, segmentation, and skeletonization. ScaffAn calculates selected parameters characterizing structural features of porcine liver scaffolds such as the sinusoidal network. After evaluating individual scaffolds, the total scores predicted scaffold interaction with cells in terms of cell adhesion. Higher scores corresponded to higher numbers of cells attached to the scaffolds. Moreover, our analysis revealed that the conventional system could not identify fine differences between good quality scaffolds while the additional use of ScaffAn allowed discrimination. This led us to the conclusion that only using the combined score resulted in the best discrimination between different quality scaffolds. Overall, our newly defined evaluation system has the potential to select the liver scaffolds most suitable for recellularization, and can represent a step toward better success in liver tissue engineering.

Experimental fortification of intestinal anastomoses with nanofibrous materials in a large animal model.

Anastomotic leakage is a severe complication in gastrointestinal surgery. It is often a reason for reoperation together with intestinal passage blockage due to formation of peritoneal adhesions. Different materials as local prevention of these complications have been studied, none of which are nowadays routinely used in clinical practice. Nanofabrics created proved to promote healing with their structure similar to extracellular matrix. We decided to study their impact on anastomotic healing and formation of peritoneal adhesions. We performed an experiment on 24 piglets. We constructed 3 hand sutured end-to-end anastomoses on the small intestine of each pig. We covered the anastomoses with a sheet of polycaprolactone nanomaterial in the first experimental group, with a sheet of copolymer of polylactic acid with polycaprolactone in the second one and no fortifying material was used in the Control group. The animals were sacrificed after 3 weeks of observation. Clinical, biochemical and macroscopic signs of anastomotic leakage or intestinal obstruction were monitored, the quality of the scar tissue was assessed histologically, and a newly developed scoring system was employed to evaluate the presence of adhesions. The material is easy to manipulate with. There was no mortality or major morbidity in our groups. No statistical difference was found inbetween the groups in the matter of level of peritoneal adhesions or the quality of the anastomoses. We created a new adhesion scoring system. The material appears to be safe however needs to be studied further to prove its' positive effects.

Allogeneic Venous Grafts of Different Origin Used for Portal Vein Reconstruction After Pancreaticoduodenectomy - Experimental Study.

BACKGROUND: In clinical medicine, little is known about the use of allografts for portal vein (PV) reconstruction after pancreaticoduodenectomy (PD). Portal and caval systems are physiologically different, therefore the properties of allografts from caval and portal systems were studied here in a pig model. MATERIALS AND METHODS: PD with PV reconstruction with allogeneic venous graft from PV or inferior vena cava (IVC) was performed in 26 pigs. Biochemical analysis and ultrasonography measurements were performed during a 4-week monitoring period. Computer simulations were used to evaluate haemodynamics in reconstructed PV and explanted allografts were histologically examined. RESULTS: The native PV and IVC grafts varied in histological structure but were able to adapt morphologically after transplantation. Computer simulation suggested PV grafts to be more susceptible to thrombosis development. Thrombosis of reconstructed PV occurred in four out of five cases in PV group. CONCLUSION: This study supports the use of allografts from caval system for PV reconstruction in clinical medicine when needed.

3D gelatin-chitosan hybrid hydrogels combined with human platelet lysate highly support human mesenchymal stem cell proliferation and osteogenic differentiation.

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin-chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin-chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin-chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%-90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin-chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin-chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.

Simultaneous Boron Ion-Channel/Growth Factor Receptor Activation for Enhanced Vascularization.

Boron ion is essential in metabolism and its concentration is regulated by ion-channel NaBC1. NaBC1 mutations cause corneal dystrophies such as Harboyan syndrome. Here a 3D molecular model for NaBC1 is proposed and it is shown that simultaneous stimulation of NaBC1 and vascular endothelial growth factor receptors (VEGFR) promotes angiogenesis in vitro and in vivo with ultralow concentrations of VEGF. Human umbilical vein endothelial cells' (HUVEC) organization into tubular structures is shown to be indicative of vascularization potential. Enhanced cell sprouting is found only in the presence of VEGF and boron, the effect abrogated after blocking NaBC1. It is demonstrated that stimulated NaBC1 promotes angiogenesis via PI3k-independent pathways and that α5 ß1 /αv ß3 integrin binding is not essential to enhanced HUVEC organization. A novel vascularization mechanism that involves crosstalk and colocalization between NaBC1 and VEGFR receptors is described. This has important translational consequences; just by administering boron, taking advantage of endogenous VEGF, in vivo vascularization is shown in a chorioallantoic membrane assay.

Engineered Coatings for Titanium Implants To Present Ultralow Doses of BMP-7.

The ongoing research to improve the clinical outcome of titanium implants has resulted in the implemetation of multiple approches to deliver osteogenic growth factors accelerating and sustaining osseointegration. Here we show the presentation of human bone morphogenetic protein 7 (BMP-7) adsorbed to titanium discs coated with poly(ethyl acrylate) (PEA). We have previously shown that PEA promotes fibronectin organization into nanonetworks exposing integrin- and growth-factor-binding domains, allowing a synergistic interaction at the integrin/growth factor receptor level. Here, titanium discs were coated with PEA and fibronectin and then decorated with ng/mL doses of BMP-7. Human mesenchymal stem cells were used to investigate cellular responses on these functionalized microenvironments. Cell adhesion, proliferation, and mineralization, as well as osteogenic markers expression (osteopontin and osteocalcin) revealed the ability of the system to be more potent in osteodifferentiation of the mesenchymal cells than combinations of titanium and BMP-7 in absence of PEA coatings. This work represents a novel strategy to improve the biological activity of titanium implants with BMP-7.

Hybrid Protein-Glycosaminoglycan Hydrogels Promote Chondrogenic Stem Cell Differentiation.

Gelatin-hyaluronic acid (Gel-HA) hybrid hydrogels have been proposed as matrices for tissue engineering because of their ability to mimic the architecture of the extracellular matrix. Our aim was to explore whether tyramine conjugates of Gel and HA, producing injectable hydrogels, are able to induce a particular phenotype of encapsulated human mesenchymal stem cells without the need for growth factors. While pure Gel allowed good cell adhesion without remarkable differentiation and pure HA triggered chondrogenic differentiation without cell spreading, the hybrids, especially those rich in HA, promoted chondrogenic differentiation as well as cell proliferation and adhesion. Secretion of chondrogenic markers such as aggrecan, SOX-9, collagen type II, and glycosaminoglycans was observed, whereas osteogenic, myogenic, and adipogenic markers (RUNX2, sarcomeric myosin, and lipoproteinlipase, respectively) were not present after 2 weeks in the growth medium. The most promising matrix for chondrogenesis seems to be a mixture containing 70% HA and 30% Gel as it is the material with the best mechanical properties from all compositions tested here, and at the same time, it provides an environment suitable for balanced cell adhesion and chondrogenic differentiation. Thus, it represents a system that has a high potential to be used as the injectable material for cartilage regeneration therapies.

Engineered microenvironments for synergistic VEGF - Integrin signalling during vascularization.

We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII12-14) and integrin binding (FNIII9-10) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications.

Vitronectin as a Micromanager of Cell Response in Material-Driven Fibronectin Nanonetworks.

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation.

Gelatin-Hyaluronic Acid Hydrogels with Tuned Stiffness to Counterbalance Cellular Forces and Promote Cell Differentiation.

Cells interact mechanically with their environment, exerting mechanical forces that probe the extracellular matrix (ECM). The mechanical properties of the ECM determine cell behavior and control cell differentiation both in 2D and 3D environments. Gelatin (Gel) is a soft hydrogel into which cells can be embedded. This study shows significant 3D Gel shrinking due to the high traction cellular forces exerted by the cells on the matrix, which prevents cell differentiation. To modulate this process, Gel with hyaluronic acid (HA) has been combined in an injectable crosslinked hydrogel with controlled Gel-HA ratio. HA increases matrix stiffness. The addition of small amounts of HA leads to a significant reduction in hydrogel shrinking after cell encapsulation (C2C12 myoblasts). We show that hydrogel stiffness counterbalanced traction forces of cells and this was decisive in promoting cell differentiation and myotube formation of C2C12 encapsulated in the hybrid hydrogels.

Living biointerfaces based on non-pathogenic bacteria support stem cell differentiation.

Lactococcus lactis, a non-pathogenic bacteria, has been genetically engineered to express the III7-10 fragment of human fibronectin as a membrane protein. The engineered L. lactis is able to develop biofilms on different surfaces (such as glass and synthetic polymers) and serves as a long-term substrate for mammalian cell culture, specifically human mesenchymal stem cells (hMSC). This system constitutes a living interface between biomaterials and stem cells. The engineered biofilms remain stable and viable for up to 28 days while the expressed fibronectin fragment induces hMSC adhesion. We have optimised conditions to allow long-term mammalian cell culture, and found that the biofilm is functionally equivalent to a fibronectin-coated surface in terms of osteoblastic differentiation using bone morphogenetic protein 2 (BMP-2) added to the medium. This living bacteria interface holds promise as a dynamic substrate for stem cell differentiation that can be further engineered to express other biochemical cues to control hMSC differentiation.