Rebesiedlung von xenogenen Gefäßmatrizen als Modell-System für eine optimierte Reendothelialisierung von parenchymatösen Gewebekonstrukten

Hintergrund: Mit Hilfe des tissue engineering (TE) und dem Verfahren der De- und Rezellularisierung wird versucht, Ersatz für geschädigte, funktionseingeschränkte Organe zu schaffen. Grundvoraussetzung für die Implantation solcher Konstrukte – und somit der Vollblutperfusion in vivo – ist eine erfolgreiche Reendothelialisierung des Gefäßsystems. Bislang wurden für die Zellbesiedlung weitestgehend Zellen von gesunden Probanden oder Labortieren verwendet. Ungeklärt ist, ob auch Endothelzellen aus vorgeschädigten Gefäßen – isoliert von Patienten mit hohem kardiovaskulärem Risikoprofil – für solche Zwecke geeignet sind. Die Reendothelialisierung dezellularisierter boviner Karotiden (BCA) mit von Patienten gewonnenen Zellen kann dabei als Modellsystem für die Entwicklung eines tissue engineerten Gefäßersatzes (TEVG) dienen. Resultierende Erkenntnisse könnten in komplexen vaskulären Systemen getestet werden. Methodik: BCA wurden ohne Zusatz von Natriumlaurylsulfat (sodium dodecyl sulfate, SDS) dezellularisiert, charakterisiert und mit endothelialen Vorläuferzellen (endothelial colony forming cells, ECFC), isoliert von Patienten mit kardiovaskulärem Risikoprofil, in Monokultur bzw. mit MSC in Kokultur reendothelialisiert. Des Weiteren wurden Rattenpankreata und -lebern dezellularisiert und mittels allogenen Langerhans Inseln rebesiedelt sowie Rattenlebern zusätzlich mit Endothelzellen (RAEC) und mesenchymalen Stromazellen (MSC) reendothelialisiert. Ergebnisse: Alle Matrices zeigten eine erfolgreiche Dezellularisierung bei erhaltener Komposition der extrazellulären Matrix. Die biomechanischen Eigenschaften der BCA waren nach Dezellularisierung weitestgehend erhalten. Trotz erfolgreicher Isolierung von ECFC von kardiovaskulär-vorerkrankten Patienten mit typischem Phänotyp war die Reendothelialiserung von BCA-Chips ausschließlich in Kokultur mit MSC erfolgreich. Die Kokultur zeigte ein konfluentes Zellwachstum, welches bei Verwendung von Ratten-MSC im Vergleich zu humanen MSC ausgeprägter war. Humane Zellen konnten bis zu 14 Tage in xenogener Zellkultur nachgewiesen werden. Parenchymatöse Organe konnten durch allogene Zellen rebesiedelt werden. Fazit: Bei Verwendung von aus Patientenblut-isolierten ECFC und Kultivierung unter statischen Bedingungen konnte nur in Kokultur mit MSC eine konfluente Endothelzellschicht ausgebildet werden. Die Rebesiedlung parenchymatöser Organe mit organtypischen sowie organfremden Zellen, die von Labortieren isoliert wurden, war erfolgreich.Background: Tissue engineering (TE) and the sub-area of decellularization and recellularization attempts to create replacements for damaged, dysfunctional organs. For implantation of such constructs - and thus whole blood perfusion in vivo - a successful reendothelialization of the vascular system is inevitable. So far, cells from healthy volunteers or laboratory animals have largely been used for recellularization. It is unclear whether endothelial cells from previously damaged vessels - isolated from patients with a high cardiovascular risk profile - can also be used for such purposes. The reendothelialization of decellularized bovine carotid arteries (BCA) with patient-derived cells can serve as a model system for the development of a tissue-engineered vascular graft (TEVG). Resulting findings could then be tested in complex vascular systems. Methods: Bovine carotid arteries (BCA) were decellularized through a novel protocol without usage of sodium dodecyl sulfate (SDS), characterized and reendothelialized using endothelial progenitor cells (ECFC), isolated from patients with cardiovascular risk factors, in monoculture or coculture with MSC. Rat pancreata and livers were de- and recellularized using allogenic islets of Langerhans while rat livers were also reendothelialized using allogenic endothelial (RAEC) and mesenchymal stromal cells (MSC). Results: All matrices showed successful decellularization while composition of extracellular matrix was retained. Biomechanical properties of decellularized BCA remained largely intact. While ECFC isolation form cardiovascular risk patients was successful and showed a typical ECFC-phenotype, recellularization of BCA-chips could only be achieved in coculture with MSC with a tendency for more confluent results using rat MSC compared to human MSC. Human cells were detectable for up to 14 days in xenogeneic cell culture. Recellularization of decellularized parenchymal organs with allogenic cells was successful. Conclusion: Upon usage of ECFC isolated from hospitalized patients under static conditions, no confluent endothelial layer was detected. A tendency for better reendothelialization in coculture with rat MSC or human MSC could be observed. Recellularization of decellularized parenchymal organs using cells from young and healthy laboratory animals could be achieved even in matrices from different organs

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

Anisotropic Brownian motion in ordered phases of DNA fragments

Using Fluorescence Recovery After Photobleaching, we investigate the Brownian motion of DNA rod-like fragments in two distinct anisotropic phases with a local nematic symmetry. The height of the measurement volume ensures the averaging of the anisotropy of the in-plane diffusive motion parallel or perpendicular to the local nematic director in aligned domains. Still, as shown in using a model specifically designed to handle such a situation and predicting a non-Gaussian shape for the bleached spot as fluorescence recovery proceeds, the two distinct diffusion coefficients of the DNA particles can be retrieved from data analysis. In the first system investigated (a ternary DNA-lipid lamellar complex), the magnitude and anisotropy of the diffusion coefficient of the DNA fragments confined by the lipid bilayers are obtained for the first time. In the second, binary DNA-solvent system, the magnitude of the diffusion coefficient is found to decrease markedly as DNA concentration is increased from isotropic to cholesteric phase. In addition, the diffusion coefficient anisotropy measured within cholesteric domains in the phase coexistence region increases with concentration, and eventually reaches a high value in the cholesteric phase