Long-term delivery of growth factors and immunomodulatory agents is highly required to support the integrity of tissue in engineering constructs, e.g., formation of vasculature, and to minimize immune response in a recipient. However, for proteins with a net positive charge at the physiological pH, controlled delivery from negatively charged alginate (Alg) platforms is challenging due to electrostatic interactions that can hamper the protein release. In order to regulate such interactions between proteins and the Alg matrix, we propose to complex proteins of interest in this study - CXCL12, FGF-2, VEGF - with polyanionic heparin prior to their encapsulation into Alg microbeads of high content of α-L-guluronic acid units (high-G). This strategy effectively reduced protein interactions with Alg (as shown by model ITC and SPR experiments) and, depending on the protein type, afforded control over the protein release for at least one month. The released proteins retained their in vitro bioactivity: CXCL12 stimulated the migration of Jurkat cells, and FGF-2 and VEGF induced proliferation and maturation of HUVECs. The presence of heparin also intensified protein biological efficiency. The proposed approach for encapsulation of proteins with a positive net charge into high-G Alg hydrogels is promising for controlled long-term protein delivery under in vivo conditions.

• MeSH
• algináty chemie   MeSH
• chemokin CXCL12 chemie   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• fibroblastový růstový faktor 2 chemie   MeSH
• heparin chemie   MeSH
• lidé MeSH
• mikrosféry MeSH
• nádorové buněčné linie MeSH
• tkáňové inženýrství MeSH
• vaskulární endoteliální růstový faktor A chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

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.

• 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

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.

• MeSH
• bodová mutace MeSH
• design s pomocí počítače * MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• fibroblastový růstový faktor 2 chemie   genetika   metabolismus   MeSH
• lidé MeSH
• počítačová simulace MeSH
• proteinové inženýrství * MeSH
• řízená evoluce molekul MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH