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Biomimetic poly(amidoamine) hydrogels as synthetic materials for cell culture.

Jacchetti E, Emilitri E, Rodighiero S, Indrieri M, Gianfelice A, Lenardi C, Podestà A, Ranucci E, Ferruti P, Milani P - J Nanobiotechnology (2008)

Bottom Line: The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls.In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip.In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.

View Article: PubMed Central - HTML - PubMed

Affiliation: Istituto di Fisiologia Generale e Chimica Biologica, Università di Milano, via Trentacoste 2, 20134 Milano, Italy. cristina.lenardi@mi.infn.it.

ABSTRACT

Background: Poly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion.

Results: A systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip.

Conclusion: The obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence analysis. Immunofluorescence analysis of actin cytoskeleton and focal contacts of TCPS or hydrogel MDCK growing cells. A) Actin stress fibers (left panel), vinculin stains (middle panel) and their merge (right panel) of MDCK cells 2 days after seeding. Small white arrows indicate short focal contacts and big white arrows indicate the long ones. In the ISA23-75 panels, red small arrows indicate filopodia red big arrows indicate lamellipodia. Nuclei are labeled with DAPI and visualized in blue. Scale bar: 10 μm. B) The percentage of cell islands with well formed stress fibers is plotted against the time after seeding. 62 (TCPS), 105 (AGMA1-75) and 72 (ISA23-75) cell islands from 3 different experiments were scored.
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Figure 12: Immunofluorescence analysis. Immunofluorescence analysis of actin cytoskeleton and focal contacts of TCPS or hydrogel MDCK growing cells. A) Actin stress fibers (left panel), vinculin stains (middle panel) and their merge (right panel) of MDCK cells 2 days after seeding. Small white arrows indicate short focal contacts and big white arrows indicate the long ones. In the ISA23-75 panels, red small arrows indicate filopodia red big arrows indicate lamellipodia. Nuclei are labeled with DAPI and visualized in blue. Scale bar: 10 μm. B) The percentage of cell islands with well formed stress fibers is plotted against the time after seeding. 62 (TCPS), 105 (AGMA1-75) and 72 (ISA23-75) cell islands from 3 different experiments were scored.

Mentions: RGD sequence from fibronectin has been shown to interact with αVβ3 integrin [12] that is expressed on MDCK cells [30]. Integrin occupancy and clustering determine the activation of a signaling pathway that ultimately affects cell adhesion, spreading and consequently cell migration, by the interaction with cytoskele-tal proteins [34,35]. Though the cell adhesion on TCPS and AGMA1-75 4 hr after seeding is similar, cell spreading is less effective on the AGMA1-75 hydrogel compared to TCPS (50% ± 14% of spread cells on AGMA1-75 and 96% ± 1% of spread cells on TCPS, n = 10). In order to determine whether AGMA1-75 hydrogels affect cytoskeleton and focal adhesion organization of MDCK cells, actin filaments and vinculin, an adhesion component, were visualized. It was found that TCPS growing cells, 24 hr after seeding, present 88% (n = 24) of cell islands with well formed stress fibers and this value decreased 48 hr after seeding to 47% (n = 38). AGMA1-75 and ISA23-75 growing cells had the opposite behavior: the number of stress fibers containing islands increased during time with a steeper increase for AGMA1-75 compare to ISA23-75 (AGMA1-75 = 21%, n = 34 and 46%, n = 71; ISA23-75 = 21%, n = 19 and 30%, n = 53, 24 hr and 48 hr after seeding, respectively, as shown in Figure 12B). TCPS and AGMA1-75 growing cells present heterogeneity in terms of length, size and thickness of vinculin-stained focal adhesion (Figure 12A). On both substrates it is possible to find short (small arrows) or long (big arrows) focal adhesions 48 hr after seeding.


Biomimetic poly(amidoamine) hydrogels as synthetic materials for cell culture.

Jacchetti E, Emilitri E, Rodighiero S, Indrieri M, Gianfelice A, Lenardi C, Podestà A, Ranucci E, Ferruti P, Milani P - J Nanobiotechnology (2008)

Immunofluorescence analysis. Immunofluorescence analysis of actin cytoskeleton and focal contacts of TCPS or hydrogel MDCK growing cells. A) Actin stress fibers (left panel), vinculin stains (middle panel) and their merge (right panel) of MDCK cells 2 days after seeding. Small white arrows indicate short focal contacts and big white arrows indicate the long ones. In the ISA23-75 panels, red small arrows indicate filopodia red big arrows indicate lamellipodia. Nuclei are labeled with DAPI and visualized in blue. Scale bar: 10 μm. B) The percentage of cell islands with well formed stress fibers is plotted against the time after seeding. 62 (TCPS), 105 (AGMA1-75) and 72 (ISA23-75) cell islands from 3 different experiments were scored.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2605745&req=5

Figure 12: Immunofluorescence analysis. Immunofluorescence analysis of actin cytoskeleton and focal contacts of TCPS or hydrogel MDCK growing cells. A) Actin stress fibers (left panel), vinculin stains (middle panel) and their merge (right panel) of MDCK cells 2 days after seeding. Small white arrows indicate short focal contacts and big white arrows indicate the long ones. In the ISA23-75 panels, red small arrows indicate filopodia red big arrows indicate lamellipodia. Nuclei are labeled with DAPI and visualized in blue. Scale bar: 10 μm. B) The percentage of cell islands with well formed stress fibers is plotted against the time after seeding. 62 (TCPS), 105 (AGMA1-75) and 72 (ISA23-75) cell islands from 3 different experiments were scored.
Mentions: RGD sequence from fibronectin has been shown to interact with αVβ3 integrin [12] that is expressed on MDCK cells [30]. Integrin occupancy and clustering determine the activation of a signaling pathway that ultimately affects cell adhesion, spreading and consequently cell migration, by the interaction with cytoskele-tal proteins [34,35]. Though the cell adhesion on TCPS and AGMA1-75 4 hr after seeding is similar, cell spreading is less effective on the AGMA1-75 hydrogel compared to TCPS (50% ± 14% of spread cells on AGMA1-75 and 96% ± 1% of spread cells on TCPS, n = 10). In order to determine whether AGMA1-75 hydrogels affect cytoskeleton and focal adhesion organization of MDCK cells, actin filaments and vinculin, an adhesion component, were visualized. It was found that TCPS growing cells, 24 hr after seeding, present 88% (n = 24) of cell islands with well formed stress fibers and this value decreased 48 hr after seeding to 47% (n = 38). AGMA1-75 and ISA23-75 growing cells had the opposite behavior: the number of stress fibers containing islands increased during time with a steeper increase for AGMA1-75 compare to ISA23-75 (AGMA1-75 = 21%, n = 34 and 46%, n = 71; ISA23-75 = 21%, n = 19 and 30%, n = 53, 24 hr and 48 hr after seeding, respectively, as shown in Figure 12B). TCPS and AGMA1-75 growing cells present heterogeneity in terms of length, size and thickness of vinculin-stained focal adhesion (Figure 12A). On both substrates it is possible to find short (small arrows) or long (big arrows) focal adhesions 48 hr after seeding.

Bottom Line: The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls.In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip.In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.

View Article: PubMed Central - HTML - PubMed

Affiliation: Istituto di Fisiologia Generale e Chimica Biologica, Università di Milano, via Trentacoste 2, 20134 Milano, Italy. cristina.lenardi@mi.infn.it.

ABSTRACT

Background: Poly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion.

Results: A systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip.

Conclusion: The obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.

No MeSH data available.


Related in: MedlinePlus