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Interaction of human plasma proteins with thin gelatin-based hydrogel films: a QCM-D and ToF-SIMS study.

Schönwälder SM, Bally F, Heinke L, Azucena C, Bulut ÖD, Heißler S, Kirschhöfer F, Gebauer TP, Neffe AT, Lendlein A, Brenner-Weiß G, Lahann J, Welle A, Overhage J, Wöll C - Biomacromolecules (2014)

Bottom Line: This technique enables the determination of adsorbant mass and changes in the shear modulus of the hydrogel layer upon adsorption of human proteins.Furthermore, Secondary Ion Mass Spectrometry and principal component analysis was applied to monitor the changed composition of the topmost adsorbate layer.This approach opens interesting perspectives for a sensitive screening of viscoelastic biomaterials that could be used for regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces (IFG) , 76344 Eggenstein-Leopoldshafen, Germany.

ABSTRACT
In the fields of surgery and regenerative medicine, it is crucial to understand the interactions of proteins with the biomaterials used as implants. Protein adsorption directly influences cell-material interactions in vivo and, as a result, regulates, for example, cell adhesion on the surface of the implant. Therefore, the development of suitable analytical techniques together with well-defined model systems allowing for the detection, characterization, and quantification of protein adsorbates is essential. In this study, a protocol for the deposition of highly stable, thin gelatin-based films on various substrates has been developed. The hydrogel films were characterized morphologically and chemically. Due to the obtained low thickness of the hydrogel layer, this setup allowed for a quantitative study on the interaction of human proteins (albumin and fibrinogen) with the hydrogel by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). This technique enables the determination of adsorbant mass and changes in the shear modulus of the hydrogel layer upon adsorption of human proteins. Furthermore, Secondary Ion Mass Spectrometry and principal component analysis was applied to monitor the changed composition of the topmost adsorbate layer. This approach opens interesting perspectives for a sensitive screening of viscoelastic biomaterials that could be used for regenerative medicine.

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Fabricationof thin gelatin-based hydrogel films: chemical structures(a) and fabrication process of the cross-linked film (b).
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fig1: Fabricationof thin gelatin-based hydrogel films: chemical structures(a) and fabrication process of the cross-linked film (b).

Mentions: To obtain gelatin-coatedquartz sensors for subsequent proteinadsorption measurements, we developed a new protocol inspired by thesynthesis reported for the production of thick gelatin hydrogels (macroscale)by Tronci.20 Briefly, a cross-linking reactionwas performed between free amine groups of individual gelatin chainsand LDI, a bifunctional diisocyanate, forming urea bonds. Due to possiblehydrolysis of the diisocyanate, also oligomeric cross-links and graftedside chains are likely formed, see Figure 1. To fabricate thin hydrogel films (nanoscale), required for QCM-Dstudies, a diluted aqueous solution of gelatin was spin-coated ontothe substrate and the obtained coating was then dipped into the LDIsolution to cross-link gelatin. Depending on the nature of the substrateand on subsequent operating conditions (biological environment), covalentbonding of the hydrogel to the substrate may be necessary. The adhesionof the hydrogel film was guaranteed by prefunctionalizing the substratevia chemical vapor deposition (CVD) polymerization. Poly(4-aminomethyl-p-xylylene-co-p-xylylene)was deposited on the substrate to obtain a reactive polymer layer,providing free amine groups (ToF-SIMS analysis, see Supporting Information, Figure SI-1). As measured by ellipsometry,a 15 to 20 nm thick polymer coating was deposited on the substrateby CVD polymerization. Subsequently, spin-coating of a 1 wt % gelatinsolution deposited a hydrogel film of 15 ± 1 nm thickness (drystate). Cross-linking with LDI enabled the immobilization of gelatinon the CVD substrate (Figure 1). Even afterwashing, the cross-linked gelatin-based hydrogel film remained onthe substrate. Hydrogel film thickness in the hydrated state is estimatedto 35–40 nm, which corresponds to a degree of swelling of 230–270vol %. In contrast, non-cross-linked gelatin films were removed bywashing (remaining thickness of 1.5 ± 1 nm on the CVD coating,and remaining thickness close to zero on pure silicon substrate).Exposing the cross-linked gelatin layers to a 10 M urea solution containing0.1% SDS, pH 3.4, resulted in no film thickness decrease, as monitoredby QCM-D and ex situ ellipsometry. Indentation by AFM was used todetermine the reduced elastic modulus Er. Values of Er = 27 ± 3 kPa to 174± 51 kPa were determined at 25 °C, depending on the indentationforce (8 nN to 1 nN). Lower Er valueswere determined at 37 °C (18 ± 2 to 106 ± 19 kPa),which can be rationalized by a stabilization of the hydrogel throughtriple helical regions of the gelatin, which disentangle upon heating.This corresponds to the behavior of bulk materials received from LDIcross-linking of gelatin. Furthermore, the mechanical data corroboratethe cross-linking in addition to the grafting of the gelatin to thesubstrate, as in the latter case at 37 °C much lower Er values would have been anticipated.


Interaction of human plasma proteins with thin gelatin-based hydrogel films: a QCM-D and ToF-SIMS study.

Schönwälder SM, Bally F, Heinke L, Azucena C, Bulut ÖD, Heißler S, Kirschhöfer F, Gebauer TP, Neffe AT, Lendlein A, Brenner-Weiß G, Lahann J, Welle A, Overhage J, Wöll C - Biomacromolecules (2014)

Fabricationof thin gelatin-based hydrogel films: chemical structures(a) and fabrication process of the cross-linked film (b).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215905&req=5

fig1: Fabricationof thin gelatin-based hydrogel films: chemical structures(a) and fabrication process of the cross-linked film (b).
Mentions: To obtain gelatin-coatedquartz sensors for subsequent proteinadsorption measurements, we developed a new protocol inspired by thesynthesis reported for the production of thick gelatin hydrogels (macroscale)by Tronci.20 Briefly, a cross-linking reactionwas performed between free amine groups of individual gelatin chainsand LDI, a bifunctional diisocyanate, forming urea bonds. Due to possiblehydrolysis of the diisocyanate, also oligomeric cross-links and graftedside chains are likely formed, see Figure 1. To fabricate thin hydrogel films (nanoscale), required for QCM-Dstudies, a diluted aqueous solution of gelatin was spin-coated ontothe substrate and the obtained coating was then dipped into the LDIsolution to cross-link gelatin. Depending on the nature of the substrateand on subsequent operating conditions (biological environment), covalentbonding of the hydrogel to the substrate may be necessary. The adhesionof the hydrogel film was guaranteed by prefunctionalizing the substratevia chemical vapor deposition (CVD) polymerization. Poly(4-aminomethyl-p-xylylene-co-p-xylylene)was deposited on the substrate to obtain a reactive polymer layer,providing free amine groups (ToF-SIMS analysis, see Supporting Information, Figure SI-1). As measured by ellipsometry,a 15 to 20 nm thick polymer coating was deposited on the substrateby CVD polymerization. Subsequently, spin-coating of a 1 wt % gelatinsolution deposited a hydrogel film of 15 ± 1 nm thickness (drystate). Cross-linking with LDI enabled the immobilization of gelatinon the CVD substrate (Figure 1). Even afterwashing, the cross-linked gelatin-based hydrogel film remained onthe substrate. Hydrogel film thickness in the hydrated state is estimatedto 35–40 nm, which corresponds to a degree of swelling of 230–270vol %. In contrast, non-cross-linked gelatin films were removed bywashing (remaining thickness of 1.5 ± 1 nm on the CVD coating,and remaining thickness close to zero on pure silicon substrate).Exposing the cross-linked gelatin layers to a 10 M urea solution containing0.1% SDS, pH 3.4, resulted in no film thickness decrease, as monitoredby QCM-D and ex situ ellipsometry. Indentation by AFM was used todetermine the reduced elastic modulus Er. Values of Er = 27 ± 3 kPa to 174± 51 kPa were determined at 25 °C, depending on the indentationforce (8 nN to 1 nN). Lower Er valueswere determined at 37 °C (18 ± 2 to 106 ± 19 kPa),which can be rationalized by a stabilization of the hydrogel throughtriple helical regions of the gelatin, which disentangle upon heating.This corresponds to the behavior of bulk materials received from LDIcross-linking of gelatin. Furthermore, the mechanical data corroboratethe cross-linking in addition to the grafting of the gelatin to thesubstrate, as in the latter case at 37 °C much lower Er values would have been anticipated.

Bottom Line: This technique enables the determination of adsorbant mass and changes in the shear modulus of the hydrogel layer upon adsorption of human proteins.Furthermore, Secondary Ion Mass Spectrometry and principal component analysis was applied to monitor the changed composition of the topmost adsorbate layer.This approach opens interesting perspectives for a sensitive screening of viscoelastic biomaterials that could be used for regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces (IFG) , 76344 Eggenstein-Leopoldshafen, Germany.

ABSTRACT
In the fields of surgery and regenerative medicine, it is crucial to understand the interactions of proteins with the biomaterials used as implants. Protein adsorption directly influences cell-material interactions in vivo and, as a result, regulates, for example, cell adhesion on the surface of the implant. Therefore, the development of suitable analytical techniques together with well-defined model systems allowing for the detection, characterization, and quantification of protein adsorbates is essential. In this study, a protocol for the deposition of highly stable, thin gelatin-based films on various substrates has been developed. The hydrogel films were characterized morphologically and chemically. Due to the obtained low thickness of the hydrogel layer, this setup allowed for a quantitative study on the interaction of human proteins (albumin and fibrinogen) with the hydrogel by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). This technique enables the determination of adsorbant mass and changes in the shear modulus of the hydrogel layer upon adsorption of human proteins. Furthermore, Secondary Ion Mass Spectrometry and principal component analysis was applied to monitor the changed composition of the topmost adsorbate layer. This approach opens interesting perspectives for a sensitive screening of viscoelastic biomaterials that could be used for regenerative medicine.

Show MeSH
Related in: MedlinePlus