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Published in Splinter Robert, Illustrated Encyclopedia of Applied and Engineering Physics, 2017
[biomedical, chemical, mechanics, signal] A 68 kDa neurofilament antibody cytoskeletal signal transduction adaptor protein involved in cellular organization as well as functioning of focal cellular adhesions. Paxillin adaptor proteins are critical in cell adhesion as well as in cellular migration.
A preliminary study on surface bioactivation of polyaryletherketone by UV-grafting with PolyNaSS: influence on osteogenic and antibacterial activities
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Yijin Wang, Yabing Jin, Yiyi Chen, Tianlei Han, Yuhong Chen, Chen Wang
RT-PCR was used to quantitatively analyze the expression of osteogenesis- and adhesion-related genes. In the analysis of osteogenesis-related genes, which included osteocalcin (OCN), osteopontin (OPN), type I collagen (Col I), runt-related transcription factor 2 (RUNX-2) and alkaline phosphatase (ALP), total RNA was isolated from hWJ-MSCs cultured on the materials in osteogenic medium for 7 and 14 d using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). In the analysis of adhesion-related genes, which included focal adhesion kinase (FAK), vinculin (VCL), paxillin (PXN), integrin β1 (ITGβ1), integrin β3 (ITGβ3) and integrin α5 (ITGα5), total RNA was isolated as described above from hWJ-MSCs cultured in common medium for 1 d. Complementary DNA (cDNA) was synthesized from 1.0 μg of total RNA using PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan) in a T3 thermocycler (Mastercycler 5333, Eppendorf, Hamburg, Germany). RT-PCR was performed using FastStart Universal SYBR Green Master Kit (Roche, Basel, Switzerland) on a real-time amplification system (7900HT Fast, Applied Biosystems, Foster City, CA, USA). The mRNA expression of the target gene was calculated by the 2−ΔΔCt method and then normalized to that of the housekeeping gene GAPDH. The primer sequences are listed in Table 2.
Preparative enrichment of human tissue cells capable to change a site of growth in vitro or in vivo - Recent developments
Published in Preparative Biochemistry and Biotechnology, 2018
Johann Bauer, Hari H. P. Cohly, Jayashree Sahana, Daniela Grimm
The observation of equal key proteins in electrophoretic studies and in experiments of exposing tissue cells to the RPM strongly suggests a common role of them in both processes (Table 1). Cell electrophoresis is based on negative charges located on cell surfaces and can be performed on cells obtained from in vitro cultures as well as on cells isolated from an organism.[21,23,26,27] The negative charges very often belong to sugar acids like the sialic acid,[22,72] which are members of whole sugar chains bound to membrane proteins.[73] They need to be located at the outermost layer of the glycocalyx in order to affect the cell’s electrophoretic mobility as it was demonstrated by tagging the cells with positively charged antibodies.[74] Repulsion forces exerted by negative charges of neighboring cells do not prevent their contact.[75] In a monolayer, the cells are bound to their neighboring cells and to an underlying sheet formed by the extracellular matrix (ECM). The ECM components are secreted by the cells immediately after seeding and bind to the bottom of a culture flask through electrostatic or/and hydrophobic forces offering an anchor place for special cell membrane receptors.[62–64] If formation of 3 D aggregates occurs scaffold-free, the cells detach from the bottom of the culture flasks.[54] During the process of detachment, the cells break their binding to the underground formed by ECM.[62–64] A well-known bridge between cells and the underground is the integrin – fibronectin system. Binding of integrins to fibronectin is regulated by sialylation of the integrin-beta 1 component.[76] Enhanced sialic acid compounds which affect the cells’ electrophoretic behavior, weaken the binding of integrin to fibronectin.[77] This influences the integrin-beta 1 association with the focal adhesion kinase, paxillin, vinculin, and other proteins,[78] whose rearrangement during 3 D aggregate formation has recently been described.[57] Hence, it may be suggested that a weakening of the cells’ binding to the ECM is responsible for the cells’ leaving a monolayer as a diminution of integrin-beta 1 has been observed in earlier studies.[18,54,58] This hypothesis is supported by an enhancement of caveolin 1 measured when spheroid formation was prevented by high cell density.[79] The latter protein forms complexes with sialidase and stabilizes the integrin binding to fibronectin.[80,81]
Astrocyte 3D culture and bioprinting using peptide functionalized hyaluronan hydrogels
Published in Science and Technology of Advanced Materials, 2023
Isabelle Matthiesen, Michael Jury, Fatemeh Rasti Boroojeni, Saskia L. Ludwig, Muriel Holzreuter, Sebastian Buchmann, Andrea Åman Träger, Robert Selegård, Thomas E. Winkler, Daniel Aili, Anna Herland
After 6 days of culture in the hydrogels, the cells were lysed, and mRNA expression levels were measured and normalized with respect to cells cultured in conventional 2D tissue culture plates. (Figures 4, 5). For FPA, five attachment- and two astrocytic-related markers were evaluated. For U87, the two astrocytic-related markers were left out in the analysis. Integrin ITGB1 was detected in both cell types, with no difference in expression for any of the hydrogel conditions or cell types (Figures 4(a), 5(a)) with p-values presented in Table S1 a) and b). The intermediate filament protein vimentin (VIM), the focal adhesion protein paxillin (PAX), and the focal adhesion kinase protein tyrosine kinase 2 (PTK2) showed no changes in expression between cells cultured in non-functionalized hydrogels and hydrogels functionalized with cRGD or IKVAV (Figures 4, 5(b-d)), see Table S1 for p-values. These findings indicate that the cells continue to probe and interact with their pericellular microenvironment also in the absence of engineered integrin-binding motifs. Previous studies have found that cells encapsulated within non-adhesive HA-based hydrogels indeed both secrete fibronectin and express integrins as a result of reciprocal cell–hydrogel interactions, which can influence the cell fate [30]. The maintained expression levels of integrins, PAX, and PTK2 in non-peptide functionalized hydrogels could thus likely be a result of cell-mediated formation of a proteinaceous pericellular matrix that would allow for integrin-mediated interactions. The role of integrin-mediated cell–hydrogel interactions was further underscored by the observed limited role of CD44. No differences in HA-binding CD44 expression were seen for any of the cell types and between the different hydrogel conditions (Figures 4(e), 5(e)). Attempts to block the dimerization of CD44 have previously been observed to be an efficient way of controlling cellular attachment in tumors, suggesting that such studies can be interesting for understanding astrocyte adhesion to the HA:PEG hydrogels used here [31,32]. We thus further investigated CD44–hydrogel interactions by blocking the receptor with both antibodies and a small molecule-compound. Interestingly, the data did not indicate any major changes in cell–hydrogel interactions (data not shown). However, a larger panel of CD44 blocking methods would be needed to fully exclude this potential mode of cell–hydrogel interaction. Furthermore, two astrocytic markers were assayed for FPA; the calcium-binding protein B (S100B) and the glial fibrillary acidic protein (GFAP). No difference in mRNA expression levels was seen upon functionalization of the hydrogels with neither IKVAV nor cRGD (Figure 4(f-g)), indicating that astrocyte phenotype was not influenced by additional integrin-mediated interactions between FPA and the hydrogels. The expression of all studied genes thus remained conserved across the 2D and 3D culture systems studied here and suggests that we have a stable in vitro phenotype in both conditions.