Neue Ansätze zur Charakterisierung von dezellularisierten Geweben und zur Rebesiedelung von Gefäßen

Die Organ bzw. Gewebe De- und Rezellularisierung stellt ein spannendes Teilgebiet des Tissue Engineering dar. Diese Habilitationsschrift setzt sich mit der tiefgreifenden Charakterisierung von dezellularisierten Geweben auseinander. Ein weiterer wichtiger Aspekt, welcher in dieser Arbeit behandelt wird ist die Reendothelialisierung von Gefäßen

Challenges of single-stage pancreatoduodenectomy: how to address pancreatogastrostomies with robotic-assisted surgery

Introduction: Establishing a sufficient pancreatico-enteric anastomosis remains one of the most important challenges in open single stage pancreatoduodenectomy as they are associated with persisting morbidity and mortality. Applicability on a robotic-assisted approach, however, even increases the requirements. With this analysis we introduce a dorsal-incision-only invagination type pancreatogastrostomy (dioPG) to the field of robotic assistance having been previously proven feasible in the field of open pancreatoduodenectomy and compare initial results to the open approach by means of morbidity and mortality. Methods: An overall of 142 consecutive patients undergoing reconstruction via the novel dioPG, 38 of them in a robotic-assisted and 104 in an open approach, was identified and further reviewed for perioperative parameters, complications and mortality. Results: We observed a comparable R0-resection rate (p = 0.448), overall complication rate (p = 0.52) and 30-day mortality (p = 0.71) in both groups. Rates of common complications, such as postoperative pancreatic fistula (p = 0.332), postoperative pancreatic hemorrhage (p = 0.242), insufficiency of pancreatogastrostomy (p = 0.103), insufficiency of hepaticojejunostomy (p = 0.445) and the re-operation rate (p = 0.103) were comparable. The procedure time for the open approach was significantly shorter compared to the robotic-assisted approach (p = 0.024). Discussion: The provided anastomosis appeared applicable to a robotic-assisted setting resulting in comparable complication and mortality rates when compared to an open approach. Nevertheless, also in the field of robotic assistance establishing a predictable pancreatico-enteric anastomosis remains the most challenging aspect of modern single-stage pancreatoduodenectomy and requires expertise and experience

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided

Thoracic Surgery in the COVID-19 Pandemic: A Novel Approach to Reach Guideline Consensus

The COVID-19 pandemic challenges international and national healthcare systems. In the field of thoracic surgery, procedures may be deferred due to mandatory constraints of the access to diagnostics, staff and follow-up facilities. There is a lack of prospective data on the management of benign and malignant thoracic conditions in the pandemic. Therefore, we derived recommendations from 14 thoracic societies to address key questions on the topic of COVID-19 in the field of thoracic surgery. Respective recommendations were extracted and the degree of consensus among different organizations was calculated. A high degree of consensus was found to temporarily suspend non-critical elective procedures or procedures for benign conditions and to prioritize patients with symptomatic or advanced cancer. Prior to hospitalization, patients should be screened for respiratory symptoms indicating possible COVID-19 infection and most societies recommended to screen all patients for COVID-19 prior to admission. There was a weak consensus on the usage of serology tests and CT scans for COVID-19 diagnostics. Nearly all societies suggested to postpone elective procedures in patients with suspected or confirmed COVID-19 and recommended constant reevaluation of these patients. Additionally, we summarized recommendations focusing on precautions in the theater and the management of chest drains. This study provides a novel approach to informed guidance for thoracic surgeons during the COVID-19 pandemic in the absence of scientific evidence-based data

Surface modification of decellularized bovine carotid arteries with human vascular cells significantly reduces their thrombogenicity

Background: Since autologous veins are unavailable when needed in more than 20% of cases in vascular surgery, the production of personalized biological vascular grafts for implantation has become crucial. Surface modification of decellularized xenogeneic grafts with vascular cells to achieve physiological luminal coverage and eventually thromboresistance is an important prerequisite for implantation. However, ex vivo thrombogenicity testing remains a neglected area in the field of tissue engineering of vascular grafts due to a multifold of reasons. Methods: After seeding decellularized bovine carotid arteries with human endothelial progenitor cells and umbilical cord-derived mesenchymal stem cells, luminal endothelial cell coverage (LECC) was correlated with glucose and lactate levels on the cell supernatant. Then a closed loop whole blood perfusion system was designed. Recellularized grafts with a LECC > 50% and decellularized vascular grafts were perfused with human whole blood for 2 h. Hemolysis and complete blood count evaluation was performed on an hourly basis, followed by histological and immunohistochemical analysis. Results: While whole blood perfusion of decellularized grafts significantly reduced platelet counts, platelet depletion from blood resulting from binding to re-endothelialized grafts was insignificant (p = 0.7284). Moreover, macroscopic evaluation revealed thrombus formation only in the lumen of unseeded grafts and histological characterization revealed lack of CD41 positive platelets in recellularized grafts, thus confirming their thromboresistance. Conclusion: In the present study we were able to demonstrate the effect of surface modification of vascular grafts in their thromboresistance in an ex vivo whole blood perfusion system. To our knowledge, this is the first study to expose engineered vascular grafts to human whole blood, recirculating at high flow rates, immediately after seeding

Perineural Invasion in Pancreatic Ductal Adenocarcinoma (PDAC): A Saboteur of Curative Intended Therapies?

(1) Background: Perineural invasion (PNI) is a common characteristic of pancreatic ductal adenocarcinoma (PDAC) and is present in most resection margins. We hypothesized that curative pancreatic tumor resection with long-term survival could only be achieved in PNI-negative patients. (2) Material and Methods: A retrospective investigation of PDAC patients who underwent curative-intended surgery during the period 2008 to 2019 was performed at our institution. (3) Results: We identified 571 of 660 (86.5%) resected patients with well-annotated reports and complete datasets. Of those, 531 patients (93%) exhibited tumors with perineural invasion (Pn1), while 40 (7%) were negative for PNI (Pn0). The majority of patients in the Pn1 group presented advanced tumor stage and positive lymph node infiltration. Patients in the Pn0 group showed an improved disease-free and long-term survival compared to the Pn1 group (p < 0.001). Subgroup analysis of all R0-resected patients indicated improved long-term survival and disease-free survival of R0 Pn0 patients when compared to R0 Pn1 patients (p < 0.001). (4) Conclusion: Our study confirmed that Pn0 improves the long-term survival of PDAC-resected cancer patients. Furthermore, PNI significantly challenges the long-term survival of formally curative (R0) resected patients. We provide new insights into the dynamics of PNI in pancreatic cancer patients which are needed to define subgroups of patients for risk stratification and multimodal treatment strategies

Hepatic artery reconstruction using an operating microscope in pediatric liver transplantation—Is it worth the effort?

Introduction: In pediatric liver transplantation (pLT), hepatic artery thrombosis (HAT) is associated with inferior transplant outcome. Hepatic artery reconstruction (HAR) using an operating microscope (OM) is considered to reduce the incidence of HAT. Methods: HAR using an OM was compared to a historic cohort using surgical loupes (SL) in pLT performed between 2009 and 2020. Primary endpoint was the occurrence of HAT. Secondary endpoints were 1-year patient and graft survival determined by Kaplan-Meier analysis and complications. Multivariate analysis was used to identify independent risk factors for HAT and adverse events. Results: A total of 79 pLTs were performed [30 (38.0%) living donations; 49 (62.0%) postmortem donations] divided into 23 (29.1%) segment 2/3, 32 (40.5%) left lobe, 4 (5.1%) extended right lobe, and 20 (25.3%) full-size grafts. One-year patient and graft survival were both 95.2% in the OM group versus 86.2% and 77.8% in the SL group (p = .276 and p = .077). HAT rate was 0% in the OM group versus 24.1% in the SL group (p = .013). One-year patient and graft survival were 64.3% and 35.7% in patient with HAT, compared to 93.9% and 92.8% in patients with no HAT (both p < .001). Multivariate analysis revealed HAR with SL (p = .022) and deceased donor liver transplantation (DDLT) (p = .014) as independent risk factors for HAT. The occurrence of HAT was independently associated with the need for retransplantation (p < .001) and biliary leakage (p = .045). Conclusion: In pLT, the use of an OM is significantly associated to reduce HAT rate, biliary complications, and graft loss and outweighs the disadvantages of delayed arterial perfusion and prolonged warm ischemia time (WIT)

technical refinement to improve the homogeneity of the decellularization process.

Hintergrund: Der Organmangel ist nach wie vor der limitierende Faktor in der klinischen Lebertransplantation. Das Konzept der De- und Rezellularisierung von parenchymatösen Organen könnte langfristig die Generierung, autologer und funktioneller Neo-Lebern ermöglichen. Dies soll durch die Rebesiedlung von dezellularisierten porcinen Lebermatrizes mit hepatisch differenzierten, induzierten, pluripotenten Stammzellen erreicht werden. Das Ziel unserer Untersuchung war die Optimierung der Perfusionshomogenität, um dadurch die Schäden der Dezellularisierung zu minimieren. Methoden: Es wurde ein bezüglich der Prozessdauer (Gesamtzeit: 7 h) und Effektivität optimiertes Dezellularisierungsprotokoll für Schweinelebern entwickelt. Dies konnte durch die Anwendung einer Druck-kontrollierten Perfusion über die Leberarterie (120mmHg) und die Pfortader (60mmHg) mit 1% Triton X-100 und 1% Natriumlaurylsulfat erreicht werden. Es wurde der Einfluss oszillierender Umgebungsdruckschwankungen auf den Dezellularisierungsprozess von Schweinelebern (n=19) untersucht. Das eigens für die Erzeugung oszillierender Umgebungsdruckschwankungen entwickelte Gerät imitiert die intraabdominellen Bedingungen während der Respiration, um die Mikroperfusion der Leber zu optimieren und dadurch die Homogenität der Dezellularisierung zu verbessern. Wir analysierten die Effektivität der Perfusionsdezellularisierung mittels makroskopischer Beobachtung, histologischer Färbungen (Hämatoxylin und Eosin [H&amp;E;], Sirius - Rot und Alcian Blau), immunohistologischer Färbungen (Kollagen IV, Laminin und Fibronektin) und biochemischer Beurteilung (DNA-, Kollagen- und Glycosaminoglykangehalt) der dezellularisierten Lebermatrizes. Die Integrität der extrazellulären Matrix (EZM) nach der Dezellularisierung wurde mit Hilfe von Ausguss-Präparaten, sowie der 3D Rekonstruktion einer computertomographischen Untersuchung der Matrix analysiert. Ergebnisse: Es konnte gezeigt werden, dass Lebern, welche mit oszillierenden Umgebungsdruckschwankungen (P(+)) perfundiert wurden, homogener dezellularisiert werden und weniger DNA-Gehalt aufweisen, verglichen mit Lebern welche ohne oszillierende Druckschwankungen (P(-)) perfundiert wurden. Mikroskopisch zeigten Lebern aus der P(-) Gruppe verbliebene Zellnester, während in Lebern der P(+) Gruppe keine Zellen in der EZM mehr sichtbar waren. Der Grad der EZM-Destruktion war höher in Lebern der P(-) Gruppe, obwohl sich die Perfusionsraten und Perfusionsdrücke nicht signifikant zwischen den Gruppen unterschieden. Die immunohistochemischen Färbungen zeigten, dass die wichtigen EZM-Komponenten nach dem Dezellularisierungsprozess erhalten bleiben. Die Ausgusspräparate zeigten ein intaktes Proteingerüst des ehemaligen Gefäßsystems (Pfortader, Leberarterie und Lebervenen), sowie ein erhaltenes Gallengangsproteinnetz. Zusammenfassung: Das hier vorgestellte Protokoll zur Dezellularisierung von porcinen Lebern ist im Vergleich zu bestehenden Protokollen schneller (7h) und effektiver. Die Anwendung oszillierender Umgebungsdruckschwankungen verbessert die Perfusionshomogenität und daraus resultierend das Ergebnis des Dezellularisierungsprozesses.Background: Organ scarcity is the major limiting factor in clinical liver transplantation programs. Decellularization and recellularization of parenchymal organs may facilitate the generation of autologous functional liver like-organs by repopulation of decellularized porcine liver matrices with induced liver cells. The aim of this study was to optimize the perfusion homogeneity in order to minimize matrix damages caused by the decellularization process. Methods: An accelerated (7 h overall perfusion time) and effective protocol for human-scale liver decellularization by pressure-controlled perfusion with 1% Triton X-100 and 1% sodium dodecyl sulfate via the hepatic artery (120 mmHg) and portal vein (60 mmHg) was developed. Furthermore, the effect of oscillating pressure conditions on pig liver decellularization (n=19) was analyzed. The proprietary perfusion device used to generate these pressure conditions mimics intra-abdominal conditions during respiration to optimize microperfusion within livers and thus optimize the homogeneity of the decellularization process. The efficiency of perfusion decellularization was analyzed by macroscopic observation, histological staining (hematoxylin and eosin [H&amp;E;], Sirius red, and alcian blue), immunohistochemical staining (collagen IV, laminin, and fibronectin), and biochemical assessment (DNA, collagen, and glycosaminoglycans) of decellularized liver matrices. The integrity of the extracellular matrix (ECM) after decellularization was visualized by corrosion casting and three- dimensional computed tomography scanning. Results: Livers perfused under oscillating pressure conditions (P(+)) showed a more homogenous course of decellularization and contained less DNA compared with livers perfused without oscillating pressure conditions (P(-)). Microscopically, livers from the (P(-)) group showed remnant cell clusters, while no cells were found in livers from the (P(+)) group. The grade of disruption of the ECM was higher in livers from the (P(-)) group, although the perfusion rates and pressure did not significantly differ. Immunohistochemical staining revealed that important matrix components were still present after decellularization. Corrosion casting showed an intact vascular (portal vein and hepatic artery) and biliary framework. Conclusion: In summary, the presented protocol for pig liver decellularization is quick (7 h) and effective. The application of oscillating pressure conditions improves the homogeneity of perfusion and thus the outcome of the decellularization process

Inhibition of Vascular Endothelial Growth Factor Protects against the Development of Oxaliplatin-Induced Sinusoidal Obstruction Syndrome in Wild-Type but Not in CD39-Null Mice

(1) Background: Sinusoidal obstruction syndrome (SOS) after oxaliplatin-based chemotherapy is associated with unfavorable outcomes after partial hepatectomy for colorectal liver metastases (CLM). Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), may prevent SOS development. We investigated the impact of VEGF-inhibition on the development of SOS in a murine model. (2) Methods: Male wild-type and CD39-null mice received oxaliplatin, additional anti-VEGF (OxAV), or controls, and were sacrificed or subjected to major partial hepatectomy (MH). Specimen were used for histological analysis of SOS. Liver damage was assessed by plasma transaminases. The VEGF pathway was elucidated by quantitative PCR of liver tissue and protein analysis of plasma. (3) Results: Mice treated with oxaliplatin developed SOS. Concomitant anti-VEGF facilitated a reduced incidence of SOS, but not in CD39-null mice. SOS was associated with increased plasma VEGF-A and decreased hepatocyte growth factor (HGF). After OxAV treatment, VEGF-R2 was upregulated in wild-type but downregulated in CD39-null mice. Oxaliplatin alone was associated with higher liver damage after MH than in mice with concomitant VEGF-inhibition. (4) Conclusions: We established a murine model of oxaliplatin-induced SOS and provided novel evidence on the protective effect of VEGF-inhibition against the development of SOS that may be associated with changes in the pathway of VEGF and its receptor VEGF-R2