Při kardiovaskulárních operacích je často nutné používat cévní náhrady o malém průměru a tkáňové záplaty. Nejvhodnějším v tomto ohledu jsou autologní tkáně, jež je často nedostatek. Alogenní nebo umělé materiály jsou často trombogenní. Cílem projektu je vytvořit nové cévní náhrady a záplaty s využitím degradabilních kolagenních gelů vyztužených nano/mikro vlákny jež budou odlity do potřebného tvaru spolu s kmenovými buňkami. Tyto konstrukty budou remodelovány v bioreaktoru za vzniku nové tkáně. Poté bude tento konstrukt decelularizován pro eliminaci imunitní se zachováním nosné struktury. In vivo ověření v prasatech bude zahrnovat dvě operace. Při první operaci bude odebráno malé množství podkožního tuku a krev, ze kterých budou izolovány kmenové a endotelové buňky, pro autologní osídlení v bioreaktoru. Poté bude následovat druhá operace na stejném zvířeti, při které bude záplata/cévní náhrada implantována na/místo a. carotis. Po 4 týdenním klinickém monitorování budou implantáty vyjmuty a hodnocení histologicky z hlediska celkové struktury, průchodnosti a trombogenicity.; During cardiovascular surgeries are small calibre vessel grafts and vascular patches required. The most suitable in this field are autologous tissues, which are often deficient. Allogeneic or artificial materials are often thrombogenic. The aim of the project is to create vascular grafts and patches using collagen gels reinforced with nano/micro fibres, moulded into the required shape along with stem cells. These constructs will be remodelled in the bioreactor forming a new tissue. This construct will be decellularized to eliminate the immune response while retaining the support structure. In vivo verification in pigs will involve two surgeries. In the first surgery, a small amount of subcutaneous fat and blood will be collected from which stem and endothelial cells will be isolated for autologous colonization in the bioreactor. Then a second operation will be followed in which the patch/vascular replacement will be implanted onto a. Carotis. After 4 weeks of clinical monitoring, implants will be removed and histologically assessed for overall structure, patency, and thrombogenicity.

• Klíčová slova
• mesenchymal stem cells, kolagen, nanovlákna, nanofibers, decelularizace, cévní náhrady, in vivo, in vivo, collagen, mesenchymální kmenové buňky, tukové kmenové buňky, endotelizace, vascular prostheses, adipose tissue derived stem cells, , decellularization, endothelization,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

BACKGROUND: Autologous vein grafts are widely used for bypass procedures in cardiovascular surgery. However, these grafts are susceptible to failure due to vein graft disease. Our study aimed to evaluate the impact of the latest-generation FRAME external support on vein graft remodeling in a preclinical model. METHODS: We performed autologous internal jugular vein interposition grafting in porcine carotid arteries for one month. Four grafts were supported with a FRAME mesh, while seven unsupported grafts served as controls. The conduits were examined through flowmetry, angiography, macroscopy, and microscopy. RESULTS: The one-month patency rate of FRAME-supported grafts was 100% (4/4), whereas that of unsupported controls was 43% (3/7, Log-rank p = 0.071). On explant angiography, FRAME grafts exhibited significantly more areas with no or mild stenosis (9/12) compared to control grafts (3/21, p = 0.0009). Blood flow at explantation was higher in the FRAME grafts (145 ± 51 mL/min) than in the controls (46 ± 85 mL/min, p = 0.066). Area and thickness of neo-intimal hyperplasia (NIH) at proximal anastomoses were similar for the FRAME and the control groups: 5.79 ± 1.38 versus 6.94 ± 1.10 mm2, respectively (p = 0.558) and 480 ± 95 vs. 587 ± 52 μm2/μm, respectively (p = 0.401). However, in the midgraft portions, the NIH area and thickness were significantly lower in the FRAME group than in the control group: 3.73 ± 0.64 vs. 6.27 ± 0.64 mm2, respectively (p = 0.022) and 258 ± 49 vs. 518 ± 36 μm2/μm, respectively (p = 0.0002). CONCLUSIONS: In our porcine model, the external mesh FRAME improved the patency of vein-to-carotid artery grafts and protected them from stenosis, particularly in the mid regions. The midgraft neo-intimal hyperplasia was two-fold thinner in the meshed grafts than in the controls.

• Publikační typ
• časopisecké články MeSH

The bioprinting of high-concentrated collagen bioinks is a promising technology for tissue engineering and regenerative medicine. Collagen is a widely used biomaterial for bioprinting because of its natural abundance in the extracellular matrix of many tissues and its biocompatibility. High-concentrated collagen hydrogels have shown great potential in tissue engineering due to their favorable mechanical and structural properties. However, achieving high cell proliferation rates within these hydrogels remains a challenge. In static cultivation, the volume of the culture medium is changed once every few days. Thus, perfect perfusion is not achieved due to the relative increase in metabolic concentration and no medium flow. Therefore, in our work, we developed a culture system in which printed collagen bioinks (collagen concentration in hydrogels of 20 and 30 mg/mL with a final concentration of 10 and 15 mg/mL in bioink) where samples flow freely in the culture medium, thus enhancing the elimination of nutrients and metabolites of cells. Cell viability, morphology, and metabolic activity (MTT tests) were analyzed on collagen hydrogels with a collagen concentration of 20 and 30 mg/mL in static culture groups without medium exchange and with active medium perfusion; the influence of pure growth culture medium and smooth muscle cells differentiation medium was next investigated. Collagen isolated from porcine skins was used; every batch was titrated to optimize the pH of the resulting collagen to minimize the difference in production batches and, therefore, the results. Active medium perfusion significantly improved cell viability and activity in the high-concentrated gel, which, to date, is the most limiting factor for using these hydrogels. In addition, based on SEM images and geometry analysis, the cells remodel collagen material to their extracellular matrix.

• Publikační typ
• časopisecké články MeSH

It is believed that 3D bioprinting will greatly help the field of tissue engineering and regenerative medicine, as live patient cells are incorporated into the material, which directly creates a 3D structure. Thus, this method has potential in many types of human body tissues. Collagen provides an advantage, as it is the most common extracellular matrix present in all kinds of tissues and is, therefore, very natural for cells and the organism. Hydrogels with highly concentrated collagen make it possible to create 3D structures without additional additives to crosslink the polymer, which could negatively affect cell proliferation and viability. This study established a new method for preparing highly concentrated collagen bioinks, which does not negatively affect cell proliferation and viability. The method is based on two successive neutralizations of the prepared hydrogel using the bicarbonate buffering mechanisms of the 2× enhanced culture medium and pH adjustment by adding NaOH. Collagen hydrogel was used in concentrations of 20 and 30 mg/mL dissolved in acetic acid with a concentration of 0.05 and 0.1 wt.%. The bioink preparation process is automated, including colorimetric pH detection and adjustment. The new method was validated using bioprinting and subsequent cultivation of collagen hydrogels with incorporated stromal cells. After 96 h of cultivation, cell proliferation and viability were not statistically significantly reduced.

• Publikační typ
• časopisecké články MeSH

Bioprinting is a modern tool suitable for creating cell scaffolds and tissue or organ carriers from polymers that mimic tissue properties and create a natural environment for cell development. A wide range of polymers, both natural and synthetic, are used, including extracellular matrix and collagen-based polymers. Bioprinting technologies, based on syringe deposition or laser technologies, are optimal tools for creating precise constructs precisely from the combination of collagen hydrogel and cells. This review describes the different stages of bioprinting, from the extraction of collagen hydrogels and bioink preparation, over the parameters of the printing itself, to the final testing of the constructs. This study mainly focuses on the use of physically crosslinked high-concentrated collagen hydrogels, which represents the optimal way to create a biocompatible 3D construct with sufficient stiffness. The cell viability in these gels is mainly influenced by the composition of the bioink and the parameters of the bioprinting process itself (temperature, pressure, cell density, etc.). In addition, a detailed table is included that lists the bioprinting parameters and composition of custom bioinks from current studies focusing on printing collagen gels without the addition of other polymers. Last but not least, our work also tries to refute the often-mentioned fact that highly concentrated collagen hydrogel is not suitable for 3D bioprinting and cell growth and development.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH