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

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.

Figure 1

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

Figure 2

AFM-based 20 μm topography scan of a scratched cross-linked gelatin-based thin film on a CVD polymer (a) and recorded profile (b) along the red line.

Figure 3

IRRAS spectra of the amino-functionalized CVD polymer (1), coated with non-cross-linked gelatin after washing (2), or coated with cross-linked gelatin prior to washing (3) and after washing (4).

Figure 4

Frequency shifts (a) and dissipation shifts (b) during Fbn adsorption on CVD coating (red), bare gold (gray), and gelatin-based hydrogel (blue) at 3rd overtone (f = 15 MHz) at 37 °C in the QCM-D study. The according frequency shift and dissipation shift over time for HSA is shown in the Supporting Information, Figure SI-2.

Figure 5

Adsorbed wet mass of Fbn on bare gold (dark gray), CVD (light gray), and gelatin film (white) calculated with the Voigt model from QCM-D measurements performed at 37 °C.

Figure 6

ΔD_n/(−Δf_n) values during the adsorption process of Fbn on the gelatin-based hydrogel (blue shades; squares), on bare gold (gray shades; circles), and on the CVD polymer (magenta shades; triangles). The corresponding ΔD_n/(−Δf_n) plot for HSA is shown in the Supporting Information, Figure SI-3. For the sake of clarity, only the third, fifth, and seventh resonance frequencies are shown.

Figure 7

Scores of PC1 (83% variance) and PC2 (12% variance) from three different experiments (n = 24), together with 95% confidence limits (a); loadings of PC1 (b); loadings of PC2 (c). For details, see SI.