Construction of a thromboresistant tissue engineered vascular graft based on de- and recellularization

Bei der peripheren arteriellen Verschlusskrankheit handelt es sich um eine durch Atherosklerose bedingte Stenose oder einen kompletten Verschluss der peripheren Arterien. In den letzten Stadien der Krankheit müssen interventionelle oder sogar offenchirurgische therapeutische Eingriffe durchgeführt werden. Im Falle der Notwendigkeit einer Bypass-Operation werden aus Gründen der Biokompatibilität und Materialeigenschaften idealerweise autologe Venen verwendet. Diese Gefäße stehen jedoch in mehr als 20 % der Fälle nicht zur Verfügung und machen die Herstellung personalisierter biologischer Gefäßtransplantate als möglichen Ersatz erstrebenswert. Die Rebesiedelung von dezellularisierten bovinen Karotiden könnte die Grundlage für ein implantierbares Gefäßtransplantat darstellen. Wesentlich für den Erfolg eines solchen Verfahrens und den Einsatz als Gefäßersatz sind dabei jedoch eine physiologische Luminalbedeckung des Gefäßtransplantats und die ex vivo Thromboresistenz. In dieser Studie wurden dezellularisierte bovine Karotiden mit humanen endothelialen Vorläuferzellen und mit aus der Nabelschnurvene isolierten humanen mesenchymalen Stammzellen rezellularisiert. Die luminale Endothelzellbedeckung (LECC) wurde anhand der Glukose- und Laktatwerte im Zellüberstand kalkuliert. Anschließend wurden rezellularisierte Gefäßtransplantate mit einer LECC >50 % und dezellularisierte Gefäßtransplantate ex vivo zwei Stunden mit humanem Vollblut perfundiert. Unter anderem wurden die Hämolysewerte und die Thrombozytenzahl stündlich ausgewertet, gefolgt von der histologischen Auswertung. Die Vollblutperfusion der dezellularisierten Gefäßtransplantate verursachte einen statistisch signifikanten Thrombozytenverbrauch. Der Thrombozytenverbrauch der mit Vollblut perfundierten rebesiedelten Gefäßtransplate war statistisch insignifikant (p = 0,7284). Dieses Ergebnis wurde durch die makroskopische Auswertung und durch die histologische Charakterisierung bestätigt, welche ergab, dass die rezellularisierten Transplantate keine CD41-positiven Thrombozyten aufwiesen. Diese Arbeit liefert somit den Nachweis für einen positiven Effekt der Oberflächenmodifikation durch Rebesiedelung von Gefäßtransplantaten auf deren Thromboresistenz in einem ex vivo Vollblutperfusionssystem.Peripheral arterial disease is stenosis or complete occlusion of the peripheral arteries due to atherosclerosis. In case of the need for bypass surgery, autologous veins are ideally used for reasons of biocompatibility and material properties. However, these vessels are not available in more than 20 % of cases, making the production of personalized biological vascular grafts as a possible substitute desirable. The repopulation of decellularized bovine carotids could provide the basis for an implantable vascular graft. We present a method to precondition vascular grafts using biological scaffolds and test thromboresistance of seeded grafts in an ex vivo setup using human whole blood. Moreover, we evaluate a non-invasive method to indicate luminal coverage (LECC) of the grafts. A 48h-protocol was established for static seeding with vascular cells, which was followed by dynamic culture over 14 (n=5) and 10 (n=9) days. Glucose and lactate were implemented as non-invasive indicators of LECC. Thromboresistance was tested in an ex vivo long-term whole blood perfusion setup. Immediately after seeding, recellularized grafts with estimated LECC >50 % were perfused for 2 hours. Perfusion of acellular scaffolds was used as a comparison. Hourly hemolysis and complete blood count evaluation was performed. Histological and immunohistochemical analysis followed. Whole blood perfusion of the decellularized vascular grafts caused a statistically significant platelet consumption. Platelet consumption of the repopulated vascular grafts perfused with whole blood was statistically insignificant (p = 0.7284). This result was confirmed by macroscopic evaluation and by histologic characterization, which revealed that the recellularized grafts had no CD41-positive platelets on the luminal surface. This study confirmed ex vivo thromboresistance of vascular grafts preconditioned with human EPC and addressed multiple bottlenecks of ex vivo dynamic whole blood perfusion experimental setups by exposing engineered vascular grafts to human whole blood recirculating at high flow rates over two hours

Rise of tissue- and species-specific 3D bioprinting based on decellularized extracellular matrix-derived bioinks and bioresins

Thanks to its natural complexity and functionality, decellularized extracellular matrix (dECM) serves as an excellent foundation for creating highly cell-compatible bioinks and bioresins. This enables the bioprinted cells to thrive in an environment that closely mimics their native ECM composition and offers customizable biomechanical properties. To formulate dECM bioinks and bioresins, one must first pulverize and/or solubilize the dECM into non-crosslinked fragments, which can then be chemically modified as needed. In bioprinting, the solubilized dECM-derived material is typically deposited and/or crosslinked in a layer-by-layer fashion to build 3D hydrogel structures. Since the introduction of the first liver-derived dECM-based bioinks, a wide variety of decellularized tissue have been employed in bioprinting, including kidney, heart, cartilage, and adipose tissue among others. This review aims to summarize the critical steps involved in tissue-derived dECM bioprinting, starting from the decellularization of the ECM to the standardized formulation of bioinks and bioresins, ultimately leading to the reproducible bioprinting of tissue constructs. Notably, this discussion also covers photocrosslinkable dECM bioresins, which are particularly attractive due to their ability to provide precise spatiotemporal control over the gelation in bioprinting. Both in extrusion printing and vat photopolymerization, there is a need for more standardized protocols to fully harness the unique properties of dECM-derived materials. In addition to mammalian tissues, the most recent bioprinting approaches involve the use of microbial extracellular polymeric substances in bioprinting of bacteria. This presents similar challenges as those encountered in mammalian cell printing and represents a fascinating frontier in bioprinting technology

Hepatitis B Immunoglobulin discontinuation in long‐term liver transplant patients

Background: Hepatitis B immunoglobulin (HBIG)-as a monotherapy or combined with nucleos(t)ide analogs (NUCs)-has effectively lowered Hepatitis B virus (HBV) reinfection after liver transplantation. However, it is associated with high costs and viral resistance. HBIG-free prophylaxis with novel NUCs (tenofovir, entecavir) composes a viable alternative. We evaluated reinfection rate, histological changes, and outcome associated with HBIG discontinuation. Methods: A retrospective analysis was performed of patients undergoing liver transplantation due to HBV-induced liver disease at our center since 1988. A controlled HBIG discontinuation was conducted between 2015 and 2017 in 65 patients. Recurrent infection was determined by HbsAg values. Fibrosis and inflammation were evaluated by routine biopsy. The survival of patients after HBIG discontinuation was compared to a control population on HBIG for prophylaxis. Results: From 1988 to 2013, 352 patients underwent liver transplantation due to HBV-induced liver disease. 169 patients could be included for analysis. 104 (51.5%) patients continued a prophylaxis containing HBIG. HBIG was discontinued in 65 (38.5%) patients in a controlled manner, maintaining an oral NUC. None of those patients showed HBV reinfection or graft dysfunction. No significant changes of inflammation grades (P = .067) or fibrosis stages (P = .051) were detected. The survival of patients after HBIG discontinuation was comparable to the control (P = .95). Conclusion: HBIG withdrawal under continuation of oral NUC therapy is safe and not related to graft dysfunction, based on blood tests and histology. HBIG-free prophylaxis is not associated with a worse outcome and displays a financial relief as well as a logistic simplification during long-term follow-up

In vitro recellularization of decellularized bovine carotid arteries using human endothelial colony forming cells

Background: Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology. Results: Decellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful. Conclusion: Using our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (< 6 mm) TEVG. ECFC isolation from the cardiovascular risk target group is possible and sufficient. Survival of diabetic ECFC appears to be highly dependent on perivascular support by rMSC/hMSC under static conditions. ECFC survival was limited to 14 days post seeding

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

Diagnostic Biomarkers to Diagnose Acute Allograft Rejection After Liver Transplantation: Systematic Review and Meta-Analysis of Diagnostic Accuracy Studies

Objective: A systematic review and meta-analysis of diagnostic biomarkers for noninvasive diagnosis of acute allograft rejection following liver transplantation.Background: Noninvasive blood and urine markers have been widely explored in recent decades for diagnosing acute rejection after liver transplantation. However, none have been translated into routine clinical use so far due to uncertain diagnostic accuracy, and liver biopsy remains the gold standard.Methods: Systematic literature searches of Medline, Cochrane and Embase were conducted up to February 2019 to identify studies evaluating the use of noninvasive markers in diagnosing allograft rejection following liver transplantation. Meta-analysis was performed using a random effects model with DerSimonian–Laird weighting and the hierarchical summary receiver operating curve.Results: Of 560 identified studies, 15 studies (1,445 patients) met the inclusion criteria. The following markers were tested: acid labile nitroso-compounds (NOx), serum amyloid A protein, procalcitonin, peripheral blood eosinophil count, peripheral blood T-cell activation and interleukin 2 (IL-2) receptor, guanylate-binding protein-2 mRNA, graft-derived cell-free DNA, pi-glutathione S-transferase, alpha-glutathione S-transferase and serum HLA class I soluble antigens. Only eosinophil count was tested in multiple studies, and they demonstrated high heterogeneity (I2 = 72% [95% CI: 0.5–0.99]). IL-2 receptor demonstrated the highest sensitivity (89% [95% CI: 0.78–0.96]) and specificity (81% [95% CI: 0.69–0.89]).Conclusion: IL-2 receptor expression demonstrated the highest diagnostic accuracy, while the peripheral eosinophil count was the only marker tested in more than one study. Presently, liver biopsy remains superior to noninvasive diagnostic biomarkers as most studies exhibited inferior designs, hindering possible translation into clinical application

The nanomolar sensing of nicotinamide adenine dinucleotide in human plasma using a cycling assay in albumin modified simulated body fluids

Abstract Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle. In particular, extracellular NAD+ has recently been demonstrated to moderate pathogenesis of multiple systemic diseases as well as aging. Herein we present an assaying method, that serves to quantify extracellular NAD+ in human heparinised plasma and exhibits a sensitivity ranging from the low micromolar into the low nanomolar domain. The assay achieves the quantification of extracellular NAD+ by means of a two-step enzymatic cycling reaction, based on alcohol dehydrogenase. An albumin modified revised simulated body fluid was employed as standard matrix in order to optimise enzymatic activity and enhance the linear behaviour and sensitivity of the method. In addition, we evaluated assay linearity, reproducibility and confirmed long-term storage stability of extracellular NAD+ in frozen human heparinised plasma. In summary, our findings pose a novel standardised method suitable for high throughput screenings of extracellular NAD+ levels in human heparinised plasma, paving the way for new clinical discovery studies

Table_1_Optimized protocol for quantification of extracellular nicotinamide adenine dinucleotide: evaluating clinical parameters and pre-analytical factors for translational research.DOCX

Nicotinamide adenine dinucleotide (NAD+), a coenzyme for more than 500 enzymes, plays a central role in energy production, metabolism, cellular signaling, and DNA repair. Until recently, NAD+ was primarily considered to be an intracellular molecule (iNAD+), however, its extracellular species (eNAD+) has recently been discovered and has since been associated with a multitude of pathological conditions. Therefore, accurate quantification of eNAD+ in bodily fluids such as plasma is paramount to answer important research questions. In order to create a clinically meaningful and reliable quantitation method, we analyzed the relationship of cell lysis, routine clinical laboratory parameters, blood collection techniques, and pre-analytical processing steps with measured plasma eNAD+ concentrations. Initially, NAD+ levels were assessed both intracellularly and extracellularly. Intriguingly, the concentration of eNAD+ in plasma was found to be approximately 500 times lower than iNAD+ in peripheral blood mononuclear cells (0.253 ± 0.02 μM vs. 131.8 ± 27.4 μM, p = 0.007, respectively). This stark contrast suggests that cellular damage or cell lysis could potentially affect the levels of eNAD+ in plasma. However, systemic lactate dehydrogenase in patient plasma, a marker of cell damage, did not significantly correlate with eNAD+ (n = 33; r = −0.397; p = 0.102). Furthermore, eNAD+ was negatively correlated with increasing c-reactive protein (CRP, n = 33; r = −0.451; p = 0.020), while eNAD+ was positively correlated with increasing hemoglobin (n = 33; r = 0.482; p = 0.005). Next, variations in blood drawing, sample handling and pre-analytical processes were examined. Sample storage durations at 4°C (0–120 min), temperature (0° to 25°C), cannula sizes for blood collection and tourniquet times (0 – 120 s) had no statistically significant effect on eNAD+ (p > 0.05). On the other hand, prolonged centrifugation (> 5 min) and a faster braking mode of the centrifuge rotor (+ levels (p + levels whereas cell damage was not correlated significantly to eNAD+ levels. The blood drawing trial did not show any influence on eNAD+, in contrast, the preanalytical steps need to be standardized for accurate eNAD+ measurement. This work paves the way towards robust eNAD+ measurements, for use in future clinical and translational research, and provides an optimized hands-on protocol for reliable eNAD+ quantification in plasma.</p