Modern tissue engineering requires not only degradable materials promoting cell growth and differentiation, but also vascularization of the engineered tissue. Porous polylactide/polycaprolactone (PLA/PCL, ratio 3/5) foam scaffolds were prepared by a combined porogen leaching and freeze-drying technique using NaCl (crystal size 250-500 µm) and a water-soluble cellulose derivative (KlucelTM E; 10-100% w/w relative to the total PLA/PCL concentration) as porogens. Scanning electron microscopy, micro-CT, and Brunauer-Emmett-Teller analysis showed that all scaffolds contained a trimodal range of pore sizes, i.e., macropores (average diameter 298-539 μm), micropores (100 nm to 10 μm), and nanopores (mostly around 3.0 nm). All scaffolds had an open porosity of about 90%, and the pores were interconnected. The size of the macropores and the nanoporosity were higher in the scaffolds prepared with Klucel. Nanoporosity increased water uptake by the scaffolds, while macroporosity promoted cell ingrowth, which was most evident in scaffolds prepared with 25% Klucel. Human adipose-derived stem cells co-cultured with endothelial cells formed pre-vascular structures in the scaffolds, which was further enhanced in a dynamic cell culture system. The scaffolds are promising for the engineering of pre-vascularized soft tissues (relatively pliable 10% Klucel scaffolds) and hard tissues (mechanically stronger 25% and 50% Klucel scaffolds).

• Klíčová slova
• compression stress and strain, degradable polyesters, dynamic cultivation, endothelial cells, macroporosity, mesenchymal stem cells, mineralization, nanoporosity, pre-vascularization, three-dimensional scaffolds,
• MeSH
• biokompatibilní materiály chemie   MeSH
• kmenové buňky cytologie   MeSH
• lidé MeSH
• polyestery * chemie   MeSH
• poréznost MeSH
• tkáňové inženýrství * metody   MeSH
• tkáňové podpůrné struktury * chemie   MeSH
• tuková tkáň cytologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biokompatibilní materiály MeSH
• poly(lactide) MeSH Prohlížeč
• polycaprolactone MeSH Prohlížeč
• polyestery * MeSH

We compared the applicability of 3D fibrous scaffolds, produced by our patented centrifugal spinning technology, in soft tissue engineering. The scaffolds were prepared from four different biocompatible and biodegradable thermoplastics, namely, polylactide (PLA), polycaprolactone (PCL), poly(3-hydroxybutyrate) (PHB), and poly(1,4-butylene succinate) (PBS) and their blends. The combined results of SEM and BET analyses revealed an internal hierarchically organized porosity of the polymeric micro/nanofibers. Both nanoporosity and capillary effect are crucial for the water retention capacity of scaffolds designed for tissue engineering. The increased surface area provided by nanoporosity enhances water retention, while the capillary effect facilitates the movement of water and nutrients within the scaffolds. When the scaffolds were seeded with adipose-derived stem cells (ASCs), the ingrowth of these cells was the deepest in the PLA/PCL 13.5/4 (w/w) composite scaffolds. This result is consistent with the relatively large pore size in the fibrous networks, the high internal porosity, and the large specific surface area found in these scaffolds, which may therefore be best suited as a component of adipose tissue substitutes that could reduce postoperative tissue atrophy. Adipose tissue constructs produced in this way could be used in the future instead of conventional fat grafts, for example, in breast reconstruction following cancer ablation.

• Klíčová slova
• PBS, PCL, PHB, PLA, bioartificial adipose tissue, centrifugal spinning technology, hierarchical inner porosity of fibers, mesenchymal stem cells, micro/nanofibrous scaffolds, tissue engineering,
• Publikační typ
• časopisecké články MeSH

Cardiovascular diseases are the most important cause of morbidity and mortality in the civilized world. Stenosis or occlusion of blood vessels leads not only to events that are directly life-threatening, such as myocardial infarction or stroke, but also to a significant reduction in quality of life, for example in lower limb ischemia as a consequence of metabolic diseases. The first synthetic polymeric vascular replacements were used clinically in the early 1950s. However, they proved to be suitable only for larger-diameter vessels, where the blood flow prevents the attachment of platelets, pro-inflammatory cells and smooth muscle cells on their inner surface, whereas in smaller-diameter grafts (6 mm or less), these phenomena lead to stenosis and failure of the graft. Moreover, these polymeric vascular replacements, like biological grafts (decellularized or devitalized), are cell-free, i.e. there are no reconstructed physiological layers of the blood vessel wall, i.e. an inner layer of endothelial cells to prevent thrombosis, a middle layer of smooth muscle cells to perform the contractile function, and an outer layer to provide innervation and vascularization of the vessel wall. Vascular substitutes with these cellular components can be constructed by tissue engineering methods. However, it has to be admitted that even about 70 years after the first polymeric vascular prostheses were implanted into human patients, there are still no functional small-diameter vascular grafts on the market. The damage to small-diameter blood vessels has to be addressed by endovascular approaches or by autologous vascular substitutes, which leads to some skepticism about the potential of tissue engineering. However, new possibilities of this approach lie in the use of modern technologies such as 3D bioprinting and/or electrospinning in combination with stem cells and pre-vascularization of tissue-engineered vascular grafts. In this endeavor, sex-related differences in the removal of degradable biomaterials by the cells and in the behavior of stem cells and pre-differentiated vascular cells need to be taken into account. Key words: Blood vessel prosthesis, Regenerative medicine, Stem cells, Footprint-free iPSCs, sr-RNA, Dynamic bioreactor, Sex-related differences.

• MeSH
• cévní protézy * MeSH
• lidé MeSH
• tkáňové inženýrství * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Scaffolds made of degradable polymers, such as collagen, polyesters or polysaccharides, are promising matrices for fabrication of bioartificial vascular grafts or patches. In this study, collagen isolated from porcine skin was processed into a gel, reinforced with collagen particles and with incorporated adipose tissue-derived stem cells (ASCs). The cell-material constructs were then incubated in a DMEM medium with 2% of FS (DMEM_part), with added polyvinylalcohol nanofibers (PVA_part sample), and for ASCs differentiation towards smooth muscle cells (SMCs), the medium was supplemented either with human platelet lysate released from PVA nanofibers (PVA_PL_part) or with TGF-β1 + BMP-4 (TGF + BMP_part). The constructs were further endothelialised with human umbilical vein endothelial cells (ECs). The immunofluorescence staining of alpha-actin and calponin, and von Willebrand factor, was performed. The proteins involved in cell differentiation, the extracellular matrix (ECM) proteins, and ECM remodelling proteins were evaluated by mass spectrometry on day 12 of culture. Mechanical properties of the gels with ASCs were measured via an unconfined compression test on day 5. Gels evinced limited planar shrinkage, but it was higher in endothelialised TGF + BMP_part gel. Both PVA_PL_part samples and TGF + BMP_part samples supported ASC growth and differentiation towards SMCs, but only PVA_PL_part supported homogeneous endothelialisation. Young modulus of elasticity increased in all samples compared to day 0, and PVA_PL_part gel evinced a slightly higher ratio of elastic energy. The results suggest that PVA_PL_part collagen construct has the highest potential to remodel into a functional vascular wall.

• Klíčová slova
• adipose tissue-derived stem cells, collagen particles, endothelial cells, extracellular matrix, gel reinforcement, remodelling, stem cells differentiation, tissue engineering, vascular patches,
• MeSH
• buněčná diferenciace MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• extracelulární matrix - proteiny metabolismus   MeSH
• gely metabolismus   MeSH
• kmenové buňky metabolismus   MeSH
• kolagen * metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• myocyty hladké svaloviny metabolismus   MeSH
• prasata MeSH
• tkáňové inženýrství metody   MeSH
• tuková tkáň * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• extracelulární matrix - proteiny MeSH
• gely MeSH
• kolagen * MeSH

Some metal nanoparticles (NP) are characterized by antimicrobial properties with the potential to be used as alternative antibiotics. However, NP may negatively impact human organism, including mesenchymal stem cells (MSC), a cell population contributing to tissue growth and regeneration. To address these issues, we investigated the toxic effects of selected NP (Ag, ZnO, and CuO) in mouse MSC. MSC were treated with various doses of NP for 4 h, 24 h, and 48 h and multiple endpoints were analyzed. Reactive oxygen species were generated after 48 h CuO NP exposure. Lipid peroxidation was induced after 4 h and 24 h treatment, regardless of NP and/or tested dose. DNA fragmentation and oxidation induced by Ag NP showed dose responses for all the periods. For other NP, the effects were observed for shorter exposure times. The impact on the frequency of micronuclei was weak. All the tested NP increased the sensitivity of MSC to apoptosis. The cell cycle was most affected after 24 h, particularly for Ag NP treatment. In summary, the tested NP induced numerous adverse changes in MSC. These results should be taken into consideration when planning the use of NP in medical applications where MSC are involved.

• Klíčová slova
• antimicrobial properties, mesenchymal stem cells, metal nanoparticles, toxicity,
• Publikační typ
• časopisecké články MeSH

Background: Cardiovascular surgery is confronted by a lack of suitable materials for patch repair. Acellular animal tissues serve as an abundant source of promising biomaterials. The aim of our study was to explore the bio-integration of decellularized or recellularized pericardial matrices in vivo. Methods: Porcine (allograft) and ovine (heterograft, xenograft) pericardia were decellularized using 1% sodium dodecyl sulfate ((1) Allo-decel and (2) Xeno-decel). We used two cell types for pressure-stimulated recellularization in a bioreactor: autologous adipose tissue-derived stromal cells (ASCs) isolated from subcutaneous fat of pigs ((3) Allo-ASC and (4) Xeno-ASC) and allogeneic Wharton’s jelly mesenchymal stem cells (WJCs) ((5) Allo-WJC and (6) Xeno-WJC). These six experimental patches were implanted in porcine carotid arteries for one month. For comparison, we also implanted six types of control patches, namely, arterial or venous autografts, expanded polytetrafluoroethylene (ePTFE Propaten® Gore®), polyethylene terephthalate (PET Vascutek®), chemically stabilized bovine pericardium (XenoSure®), and detoxified porcine pericardium (BioIntegral® NoReact®). The grafts were evaluated through the use of flowmetry, angiography, and histological examination. Results: All grafts were well-integrated and patent with no signs of thrombosis, stenosis, or aneurysm. A histological analysis revealed that the arterial autograft resembled a native artery. All other control and experimental patches developed neo-adventitial inflammation (NAI) and neo-intimal hyperplasia (NIH), and the endothelial lining was present. NAI and NIH were most prominent on XenoSure® and Xeno-decel and least prominent on NoReact®. In xenografts, the degree of NIH developed in the following order: Xeno-decel > Xeno-ASC > Xeno-WJC. NAI and patch resorption increased in Allo-ASC and Xeno-ASC and decreased in Allo-WJC and Xeno-WJC. Conclusions: In our setting, pre-implant seeding with ASC or WJC had a modest impact on vascular patch remodeling. However, ASC increased the neo-adventitial inflammatory reaction and patch resorption, suggesting accelerated remodeling. WJC mitigated this response, as well as neo-intimal hyperplasia on xenografts, suggesting immunomodulatory properties.

• Klíčová slova
• Wharton’s jelly mesenchymal stem cells, adipose tissue-derived stromal cells, allograft, blood vessel prosthesis, cell seeding, decellularization, heterograft, pericardium, tissue engineering,
• MeSH
• allogeneické buňky MeSH
• arteriae carotides MeSH
• cévní protézy MeSH
• hyperplazie MeSH
• lidé MeSH
• ovce MeSH
• perikard MeSH
• prasata MeSH
• remodelace cév * MeSH
• skot MeSH
• tkáňové inženýrství MeSH
• transplantace hematopoetických kmenových buněk * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Biological hydrogels are highly promising materials for bone tissue engineering (BTE) due to their high biocompatibility and biomimetic characteristics. However, for advanced and customized BTE, precise tools for material stabilization and tuning material properties are desired while optimal mineralisation must be ensured. Therefore, reagent-free crosslinking techniques such as high energy electron beam treatment promise effective material modifications without formation of cytotoxic by-products. In the case of the hydrogel gelatin, electron beam crosslinking further induces thermal stability enabling biomedical application at physiological temperatures. In the case of enzymatic mineralisation, induced by Alkaline Phosphatase (ALP) and mediated by Calcium Glycerophosphate (CaGP), it is necessary to investigate if electron beam treatment before mineralisation has an influence on the enzymatic activity and thus affects the mineralisation process. The presented study investigates electron beam-treated gelatin hydrogels with previously incorporated ALP and successive mineralisation via incubation in a medium containing CaGP. It could be shown that electron beam treatment optimally maintains enzymatic activity of ALP which allows mineralisation. Furthermore, the precise tuning of material properties such as increasing compressive modulus is possible. This study characterizes the mineralised hydrogels in terms of mineral formation and demonstrates the formation of CaP in dependence of ALP concentration and electron dose. Furthermore, investigations of uniaxial compression stability indicate increased compression moduli for mineralised electron beam-treated gelatin hydrogels. In summary, electron beam-treated mineralized gelatin hydrogels reveal good cytocompatibility for MG-63 osteoblast like cells indicating a high potential for BTE applications.

• Klíčová slova
• bone tissue engineering, electron beam treatment, enzymatic mineralisation, gelatin hydrogels,
• Publikační typ
• časopisecké články MeSH

Galectin-3 (Gal-3) is a β-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a β-galactoside LacdiNAc (GalNAcβ1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by β1 and αV integrins-namely α5β1, αVβ3, and αVβ1 integrins.

• Klíčová slova
• ADSC, HUVEC, carbohydrate, galectin, integrin,
• MeSH
• buněčná adheze * MeSH
• endoteliální buňky pupečníkové žíly (lidské) cytologie   fyziologie   MeSH
• galektiny metabolismus   MeSH
• integriny metabolismus   MeSH
• krevní proteiny metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   fyziologie   MeSH
• spoje buňka-matrix metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• galektiny MeSH
• integriny MeSH
• krevní proteiny MeSH
• LGALS3 protein, human MeSH Prohlížeč

Vascular endothelial growth factor-A165 (VEGF-A165) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A165 and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A165 stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A165 on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A165 have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.

• Klíčová slova
• adult stem cells, basic fibroblast growth factor (bFGF), cell adhesion, cell proliferation, endothelial cells, heterologous expression, recombinant vascular endothelial growth factor (VEGF), regenerative medicine, tissue engineering, vascular replacements,
• MeSH
• buněčná adheze účinky léků   MeSH
• endoteliální buňky pupečníkové žíly (lidské) cytologie   metabolismus   MeSH
• fibroblastový růstový faktor 2 chemie   genetika   farmakologie   MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• lidé MeSH
• prasata MeSH
• proliferace buněk účinky léků   MeSH
• rekombinantní proteiny chemie   farmakologie   MeSH
• vaskulární endoteliální růstový faktor A chemie   genetika   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH
• rekombinantní proteiny MeSH
• vaskulární endoteliální růstový faktor A MeSH
• VEGFA protein, human MeSH Prohlížeč

BACKGROUND: Adipose tissue-derived stromal cells (ADSCs) have great potential for cell-based therapies, including tissue engineering. However, various factors can influence the characteristics of isolated ADSCs. METHODS: We studied the influence of the harvesting site, i.e., inner thigh (n = 3), outer thigh (n = 3), outer thigh (n = 3), outer thigh (. RESULTS: We revealed higher initial cell yields from the outer thigh region than from the abdomen region. Negative pressure did not influence the cell yields from the outer thigh region, whereas the yields from the abdomen region were higher under high negative pressure than under low negative pressure. In the subsequent passage, in general, no significant relationship was identified between the different negative pressure and ADSC characteristics. No significant difference was observed in the characteristics of thigh ADSCs and abdomen ADSCs. Only on day 1, the diameter was significantly bigger in outer thigh ADSCs than in abdomen ADSCs. Moreover, we noted a tendency of thigh ADSCs (i.e., inner thigh+outer thigh) to reach a higher cell number on day 7. Discussion. The harvesting site and negative pressure can potentially influence initial cell yields from lipoaspirates. However, for subsequent in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.

• Publikační typ
• časopisecké články MeSH