Jaroslav Chlupac

Vascular endothelial cells on two-and three-dimensional fibrin assemblies for biomaterial coatings

Various techniques for coating synthetic surfaces with fibrin structures were tested for seeding bovine pulmonary artery endothelial cells (EC). Two-dimensional fibrin (Fb) structures (2D Fb) were obtained by successively repeating adsorption of fibrinogen (Fbg), incubating the surface with thrombin (Thr) solution, and inhibiting surface-attached Thr. Three-dimensional fibrin networks immobilized at the surface (3D Fb) were formed by catalytic action of surface-attached thrombin on an ambient Fbg solution. Ultra-thin 3D Fbs were obtained if thrombin inhibitors antithrombin III and heparin were added into an Fbg solution. The formation of surface fibrin structures was observed in situ using surface plasmon resonance. The morphology of the structures was studied by transmission and scanning electron microscopy. A polylactide fibrous scaffold was modified with a surface fibrin film without filling the inner pores with a bulk gel. The growth of EC seeded on a polystyrene surface coated with the Fb films was evaluated by the number and morphology of the adhering ECs and the concentration of beta-actin, vinculin, alpha(v)-intergrin, and von Willebrand factor (vWF). The best initial cell spreading after 1 day was observed on 2D Fb and ultra-thin 3D Fb. The highest concentration of vWF, a marker of EC differentiation, was observed after 3 days on thick 3D Fbs. The highest EC population densities after 7 days were observed on 2D Fb and thick 3D Fb.

Improved adhesion and differentiation of endothelial cells on surface-attached fibrin structures containing extracellular matrix proteins.

Currently used vascular prostheses are hydrophobic and do not allow endothelial cell (EC) adhesion and growth. The aim of this study was to prepare fibrin (Fb)-based two-dimensional (2D) and three-dimensional (3D) assemblies coated with extracellular matrix (ECM) proteins and to evaluate the EC adhesion, proliferation and differentiation on these assemblies in vitro. Coating of Fb with collagen, laminin (LM), and fibronectin (FN) was proved using the surface plasmon resonance technique. On all Fb assemblies, ECs reached higher cell densities than on polystyrene after 3 and 7 days of culture. Immunoflurescence staining showed better assembly of talin and vinculin into focal adhesion plaques, and also more apparent staining of vascular endothelial cadherin on surface-attached 3D Fb and protein-coated Fb assemblies. On these samples, ECs also contained a lower concentration of intercellular adhesion molecule-1, measured by enzyme-linked immunosorbent assay. Higher concentrations of CD31 (platelet-endothelial cell adhesion molecule-1) were found on 3D Fb coated with LM, and higher concentrations of von Willebrand factor were found on 3D Fb coated with type I collagen or LM in comparison to 2D Fb layers. The results indicate that ECM protein-coated 2D and 3D Fb assemblies can be used for versatile applications in various tissue replacements where endothelialization is desirable, for example, vascular prostheses and heart valves.

Endothelial Cell Lining of PET Vascular Prostheses: Modification with Degradable Polyester-based Copolymers and Adhesive Protein Multi-layers

Background: Bypass surgery for atherosclerosis is confronted with the absence of endothelial cells in the lumen of vascular prosthesis in humans. This imposes a risk of thrombosis. New biomaterials try to minimize surface thrombogenicity. Methods: Knitted polyethylene terephthalate (PET) vascular graft patches were impregnated with degradable polyester polymers: poly (L-lactide-co-glycolide) (PLG) or poly (L-lactide-co-glycolide-co-e-caprolactone) (PLGC). The luminal surface was coated with collagen type I (Co) to which extracellular matrix proteins laminin (LM), fibronectin (FN), or surface fibrin gel (Fb) were attached. Three types of prostheses (PET, PET–PLG and PET– PLGC) and 5 types of protein assemblies (+Co, +Co/LM, +Co/FN, +Co/Fb, +Co/Fb/FN) were fabricated. Scanning electron microscopy and measurements of the water contact angles were performed. The development of a bovine endothelial cell layer was studied in a static culture for 1 week. Results: The cells reached confluence on all PET surfaces with the highest final density on +Co/FN. Impregnation of PET with polymers made it less adhesive for cells in the following order: PET > PET–PLG > PET– PLGC. However, additional coating with the protein assemblies enhanced the endothelial cell growth, especially on fibrin-containing surfaces. Conclusion: Tri-component vascular grafts composed of PET, copolymers and cell-adhesive assemblies were fabricated. The endothelial lining on the polymer-coated grafts was promoted after modification with the protein multilayers. Artificial vascular prostheses have been made of non-degradable, non-compliant and thrombogenic materials for more than 50 years. Thus, they resemble passive tubing. Potential bio-activation by degradable materials and by introduction of living endothelial cells may approximate these materials to native artery. This work provided a method to include bio-degradable polymers into vascular graft and to facilitate the growth of cell lining via adhesive protein multilayers.

Simultaneous living donor orthotopic renal transplantation and bilateral nephrectomy for recurrent renal cell carcinoma and renal failure: case report and review of literature

Background We report the case of a 43-year-old female patient with systemic lupus erythematosus, class III lupus nephritis, with predialysis creatinine levels around 350 μmol/L (3.95 mg/dL) after partial resection of the left kidney with histologically verified papillary carcinoma in 2010. Preoperative computed tomography of the abdomen revealed a small 8 mm tumor in the left upper kidney pole. The patient was indicated for simultaneous bilateral nephrectomy and orthotopic renal transplantation with the aim to minimize invasiveness of the procedure as well as for curable tumor removal. Method The procedure was performed under the full anesthesia trough upper middle laparotomy. As the first step, bilateral transperitoneal nephrectomy was performed. The live donor surgery started in a parallel theater to shorten the cold ischemic time of the graft. The renal graft had singe vessels and ureter; it was placed into the recipient’s right orthotopic position. End-to-end anastomosis of the right renal vein and artery anastomosis were performed; ureter was anastomosed end-to-end using recipient’s ureter. Results The postoperative period was uneventful with repeatedly excellent ultrasonography check-up of the graft’s perfusion. The patient was discharged after 13 days with a good renal function of the graft (urea: 15 mmol/L, creatinine 160 μmol/L [1.80 mg/dL]). Conclusion Orthotopic renal transplantation is a technically challenging but valid alternative for patients who are unsuitable candidates for heterotopic renal transplantation or in cases where there is a clear benefit of orthotopic renal transplantation.

The Effects of the Thoracoabdominal Aortic Metabolism on the Endovascular Procedures Outcomes

Aortic metabolism is a merge of complex processes. Atherosclerotic infiltration of the aortic wall is a crucial factor when choosing an appropriate endovascular treatment modality for anatomically suitable patients. Due to the rise of endovascular treatments and placement of endovascular devices in younger patients, many physicians are starting to take a more considerable interest in this complex process. Many experimental animal models are used for elucidation of individual aspects in this area. This led to an in-depth explanation of many metabolic processes of the aortic wall. However, only a few papers are reporting on the assessment of these pathological processes in human tissues. This paper outlines some of the crucial aspects of the thoracoabdominal aortic metabolism. Final results of endovascular treatments are believed to be significantly affected by the quality of the aortic wall and the ability to predict its further changes. This includes the pathological changes and their effects on the symbiotic metabolic changes. “Patient-tailored” endovascular aortic treatments based on the aortic metabolic assessment may be able to optimise the cases outcomes. Due to the rise of endovascular treatments and placement of endovascular devices in younger patients, further research is needed to understand better aortic metabolic processes in various patient groups, including groups of patients suffering from chronic metabolic diseases. Aortic wall metabolism should be assessed with the aim to optimise the endovascular treatment outcomes.

Automated dynamic bioreactor for 2D endothelial structures

This work deals with construction of automated bioreactor that is used for 2D planar endothelial structures in optimized cultivation chamber. Designed bioreactor is controlled by industrial PC with FPGA card. It consists of proportionally controlled peristaltic pumps for cultivation media perfusion, necessary sensors and electronics for maintaining stable and repeatable operation during experiments, redundant power supply. FPGA card design gives deterministic control with implemented safety routines. Monitoring controlling software allows remote access. Endothelial cells are during experiments exposed to laminar flow at given shear stress, which helps to orient them in direction of flow and improves structure of cytoskeleton and intercellular connections. Results from cultivation experiments leads to establishing cultivation of 3D tubular structures and artificial vascular prostheses.