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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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Scores of PC1 (83% variance) and PC2 (12% variance) fromthreedifferent experiments (n = 24), together with 95%confidence limits (a); loadings of PC1 (b); loadings of PC2 (c). Fordetails, see SI.
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fig7: Scores of PC1 (83% variance) and PC2 (12% variance) fromthreedifferent experiments (n = 24), together with 95%confidence limits (a); loadings of PC1 (b); loadings of PC2 (c). Fordetails, see SI.

Mentions: Protein solutions, prepared from humanFbn or albumin in PBS, were flushed on gelatin-based hydrogel thinfilm-coated QCM-D sensors (37 °C, 1 mg/mL). Advanced chemicalcharacterization of the adsorbed protein layers on gelatin hydrogelshas been performed by ToF-SIMS. Such experiments could not be carriedout by IRRAS, since the latter would not enable the distinction betweendifferent proteins. ToF-SIMS measurements were thus performed to characterizethe topmost layer of thin gelatin-based hydrogel film (sampling depthof static SIMS is only 2–5 nm), exposed to human proteins.Under primary ion bombardment, proteins decompose yielding characteristicfragments of the amino acid side chains. Since the signal intensitiesof the amino acids fragments define a multivariate data set (for adetailed peak list, see Table SI-2 in SupportingInformation), principal component analysis was applied to reducedata dimensionality and complexity. Using principal component analysis,we can discern between pure gelatin surfaces and those loaded withFbn or albumin. Studies of Muramoto55 andTyler56 have been addressing other setsof proteins. Figure 7a shows the clusteringof the obtained data in the principal component analysis of the characteristicamino acids fragments. Whereas PC1, capturing 84% of the variance,obviously discriminates well between the pure gelatin surfaces (Figure 7a, circles) and the HSA (squares), respectively,Fbn (triangles) treated gelatin samples; PC2 (12% variance) separatesbest the HSA-treated samples. As can be seen from the correspondingloading plots, Figure 7b (see also SI), arginine (43, and 73 m/z) has strong positive loadings for PC1, glycine (30 m/z), proline (68, and 70 m/z), and lysine (84 m/z) have strong negative loadings, nota bene gelatin has negative PC1score values. These findings are in good accordance with the aminoacid compositions of gelatin (see Table SI-3 in Supporting Information)57,58 since gelatin is richin glycine and proline. It should be noted, however, that the CH4N+ fragment might also arise from other amino acidsand leucine and isoleucine both share the SIMS fragment. Therefore,this signal can be misinterpreted. Arginine, alanine, and leucineexhibit strong negative loadings in PC2, Figure 7c. Whereas HSA is indeed richer in isoleucine and leucine as Fbnand gelatin, the reasons for the predominance of arginine and alaninein PC2 are less obvious. These effects are not fully understood today;they can be due to the spatial arrangement of the detected amino acidsaffecting the sputter yields during the SIMS process (that is alsoinfluenced by the primary ion species55), or due to side effects of the protein structure affecting ionizationprobabilities.


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)

Scores of PC1 (83% variance) and PC2 (12% variance) fromthreedifferent experiments (n = 24), together with 95%confidence limits (a); loadings of PC1 (b); loadings of PC2 (c). Fordetails, see SI.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Scores of PC1 (83% variance) and PC2 (12% variance) fromthreedifferent experiments (n = 24), together with 95%confidence limits (a); loadings of PC1 (b); loadings of PC2 (c). Fordetails, see SI.
Mentions: Protein solutions, prepared from humanFbn or albumin in PBS, were flushed on gelatin-based hydrogel thinfilm-coated QCM-D sensors (37 °C, 1 mg/mL). Advanced chemicalcharacterization of the adsorbed protein layers on gelatin hydrogelshas been performed by ToF-SIMS. Such experiments could not be carriedout by IRRAS, since the latter would not enable the distinction betweendifferent proteins. ToF-SIMS measurements were thus performed to characterizethe topmost layer of thin gelatin-based hydrogel film (sampling depthof static SIMS is only 2–5 nm), exposed to human proteins.Under primary ion bombardment, proteins decompose yielding characteristicfragments of the amino acid side chains. Since the signal intensitiesof the amino acids fragments define a multivariate data set (for adetailed peak list, see Table SI-2 in SupportingInformation), principal component analysis was applied to reducedata dimensionality and complexity. Using principal component analysis,we can discern between pure gelatin surfaces and those loaded withFbn or albumin. Studies of Muramoto55 andTyler56 have been addressing other setsof proteins. Figure 7a shows the clusteringof the obtained data in the principal component analysis of the characteristicamino acids fragments. Whereas PC1, capturing 84% of the variance,obviously discriminates well between the pure gelatin surfaces (Figure 7a, circles) and the HSA (squares), respectively,Fbn (triangles) treated gelatin samples; PC2 (12% variance) separatesbest the HSA-treated samples. As can be seen from the correspondingloading plots, Figure 7b (see also SI), arginine (43, and 73 m/z) has strong positive loadings for PC1, glycine (30 m/z), proline (68, and 70 m/z), and lysine (84 m/z) have strong negative loadings, nota bene gelatin has negative PC1score values. These findings are in good accordance with the aminoacid compositions of gelatin (see Table SI-3 in Supporting Information)57,58 since gelatin is richin glycine and proline. It should be noted, however, that the CH4N+ fragment might also arise from other amino acidsand leucine and isoleucine both share the SIMS fragment. Therefore,this signal can be misinterpreted. Arginine, alanine, and leucineexhibit strong negative loadings in PC2, Figure 7c. Whereas HSA is indeed richer in isoleucine and leucine as Fbnand gelatin, the reasons for the predominance of arginine and alaninein PC2 are less obvious. These effects are not fully understood today;they can be due to the spatial arrangement of the detected amino acidsaffecting the sputter yields during the SIMS process (that is alsoinfluenced by the primary ion species55), or due to side effects of the protein structure affecting ionizationprobabilities.

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