Thrombogenicity assessment of perfusable tissue engineered constructs: a systematic review.

Vascular surgery faces a critical demand for novel vascular grafts that are biocompatible and thromboresistant. This urgency particularly applies to bypass operations involving small caliber vessels. In the realm of tissue engineering, the development of fully vascularized organs holds great promise as a solution to organ shortage for transplantation. To achieve this, it is imperative to (re-)construct a biocompatible and non-thrombogenic vascular network within these organs. In this systematic review, we identify, classify and discuss basic principles and methods used to perform in vitro/ex vivo dynamic thrombogenicity testing of perfusable tissue engineered organs and tissues. We conducted a pre-registered systematic review of studies published in the last 23 years according to PRISMA-P Guidelines, comprising a systematic data extraction, in-depth analysis and risk of bias assessment of 116 included studies. We identified shaking (n=28), flow loop (n=17), ex vivo (arterio-venous shunt, n=33) and dynamic in vitro models (n=38) as main approaches for thrombogenicity assessment. This comprehensive review unveils a prevalent lack of standardization and serves as a valuable guide in the design of standardized experimental setups.

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.

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.

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.

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.