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. 2014 Aug 5;9(8):e104019.
doi: 10.1371/journal.pone.0104019. eCollection 2014.

In vitro study of a novel nanogold-collagen composite to enhance the mesenchymal stem cell behavior for vascular regeneration

Huey-Shan Hung  1 , Chih-Hsuan Chang  2 , Chen-Jung Chang  3 , Cheng-Ming Tang  4 , Wei-Chien Kao  2 , Shinn-Zong Lin  5 , Hsien-Hsu Hsieh  6 , Mei-Yun Chu  2 , Wei-Shen Sun  2 , Shan-Hui Hsu  7
Affiliations

In vitro study of a novel nanogold-collagen composite to enhance the mesenchymal stem cell behavior for vascular regeneration

Huey-Shan Hung et al. PLoS One. .

Abstract

Novel nanocomposites based on type I collagen (Col) containing a small amount (17.4, 43.5, and 174 ppm) of gold nanoparticles (AuNPs, approximately 5 nm) were prepared in this study. The pure Col and Col-AuNP composites (Col-Au) were characterized by the UV-Vis spectroscopy (UV-Vis), surface-enhanced raman spectroscopy (SERS) and atomic force microscopy (AFM). The interaction between Col and AuNPs was confirmed by infrared (IR) spectra. The effect of AuNPs on the biocompatibility of Col, evaluated by the proliferation and reactive oxygen species (ROS) production of mesenchymal stem cells (MSCs) as well as the activation of monocytes and platelets, was investigated. Results showed that Col-Au had better biocompatibility than Col. Upon stimulation by vascular endothelial growth factor (VEGF) and stromal derived factor-1α (SDF-1α), MSCs expressed the highest levels of αvβ3 integrin/CXCR4, focal adhesion kinase (FAK), matrix metalloproteinase-2 (MMP-2), and Akt/endothelial nitric oxide synthase (eNOS) proteins when grown on the Col-Au (43.5 ppm) nanocomposite. Taken together, Col-Au nanocomposites may promote the proliferation and migration of MSCs and stimulate the endothelial cell differentiation. These results suggest that Col-Au may be used to construct tissue engineering scaffolds for vascular regeneration.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Materials Characterization.
(a) UV–Vis absorption spectra for Col solution ater loading of different concentrations of AuNPs (∼17.4 ppm, 43.5 ppm, and 174 ppm). (b) IR spectra of Col and Col-Au nanocomposites in the total wavenumber ranges from 400 cm−1 to 4000 cm−1 region and from 1200 cm−1 to 1700 cm−1. All results are representative of one of three independent experiments. (c) AFM topography diagrams for the pure Col, and Col-Au nanocomposites containing 17.4 ppm, 43.5 ppm, and 174 ppm of AuNPs. All results are representative of one of three independent experiments. Ra is the average roughness of the sample. Scale bar = 100 nm.
Figure 2
Figure 2. Spectra of Col and Col-Au with peaks in the amide region by surface-enhanced raman spectroscopy (SERS).
Figure 3
Figure 3. Cytoskeleton and cell morphology.
(a) Rhodamine phalloidin staining for the cytoskeletal fibers of MSCs on pure Col and different Col-Au nanocomposites at 8 h and 48 h by fluorescence microscopy. Scale bar = 50 µm. (b) SEM images for MSCs on pure Col and different Col-Au nanocomposites at 48 h. Arrows indicate filopodia (green color) and lamellipodia (red color). Scale bar = 50 µm.
Figure 4
Figure 4. The proliferation of MSCs and reactive oxygen species (ROS) generation assay on different materials after 48 h of incubation.
(a) MSCs proliferation examined by MTT assay on control (TCPS), pure Col, and Col-Au nanocomposites containing 17.4 ppm, 43.5 ppm, and 174 ppm of AuNPs after 48 h of incubation. **p<0.01: greater than control (TCPS). (b) The intracellular ROS quantified by 2, 7-dichlorofluorescein diacetate (DCFH-dA) and flow cytometric analysis. **p<0.01: greater than control (TCPS).
Figure 5
Figure 5. The expression of CD68 for macrophages on different materials at 96 h.
(a) Cells were immunostained by the primary anti-CD68 antibody and conjugated with FITC-immunoglobulin secondary antibody (green color fluorescence). Cell nuclei were staiend by DAPI (blue color fluorescence). Scale bar = 50 µm. (b) CD68 expression was quantified based on fluorescence intensity. **p<0.01: smaller than control (TCPS).
Figure 6
Figure 6. SEM images showing the adhesion and activation of human blood platelets on different matierals.
Figure 7
Figure 7. The CD31 protein expression of MSCs on different materials at 3, 5, and 7 days.
(a) MSCs were immunostained by the primary anti-CD31 antibody and conjugated with FITC-immunoglobulin secondary antibody (green color fluorescence) and cell nuclear staining was performed by DAPI (blue color staining). Results were recorded by fluorescence microscopy. Scale bar = 10 µm. (b) Semi-quantitative measurement of fluorescence intensity revealed a significantly higher level of CD31 expression on Col-Au 43.5 ppm. Data are mean ± SD (n = 3). *p<0.01: greater than the other groups [including the control (TCPS), Col, and Col-Au nanocomposites at the other concentrations]; **p<0.01: greater than TCPS and Col.
Figure 8
Figure 8. The expression of αVβ3 integrin and CXCR4 for MSCs on different materials at 48 h of incubation and for those treated with either VEGF (50 ng/ml) or SDF-1α (50 ng/ml) in culture media.
(a) MSCs were immunostained by the primary anti-αvβ3 integrin antibody and primary anti-CXCR4 antibody and conjugated with FITC-immunoglobulin secondary antibody (green color fluorescence), Cy5.5-conjugated immunoglobulin secondary antibody (red color fluorescence). Cell nuclei was stained by DAPI. Scale bar = 100 µm. (b) αvβ3 integrin and CXCR4 expressions were quantified based on fluorescence intensity. *p<0.05, **p<0.01: greater than control (TCPS).
Figure 9
Figure 9. The expression of p-FAK protein and MMP-2 enzymatic activity in MSCs cultured on different materials for 48 h.
(a) Relative expression ratios of p-FAK was normalized to total FAK. **p<0.01. (b) The MMP-2 enzymatic activities was normalized to the protein content. Semi-quantitative measurement of the optical density (OD) of gelatinolytic bands revealed significantly greater MMP-2 expression for MSCs on Col-Au 43.5 ppm. *p<0.05: greater than control (TCPS).
Figure 10
Figure 10. The expression of p-Akt and eNOS proteins in MSCs cultured on different materials for 48 h by western blots and immunofluorescence staining.
(a) Relative expression ratios of p-Akt was normalized to total Akt. **p<0.01. (b) The expression of eNOS protein for MSCs was examined by fluorescence microscopy. Cells were stained with primary anti-eNOS antibody followed by FITC-conjugated immunoglobulin (green color fluorescence). Cell nuclei was stained by DAPI. Scale bar = 10 µm. Semi-quantitative measurement of fluorescence intensity revealed a significantly higher level of eNOS expression compared with the control. *p<0.01: greater than control (TCPS).
Figure 11
Figure 11. Migration of MSCs on different materials during a period of 48 h.
(a) Cell migration into the gap zone area was monitored by fluorescence microscopy. Cells were stained by calcein-AM (2 µM) prior to examination. Scale bar = 500 µm. **p<0.01: greater than control (TCPS). (b) MSCs proliferation examined by MTT assay. **p<0.01: greater than control (TCPS).
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The work was supported by both National Science Council, Taiwan, and Blood Bank, Taichung Veterans General Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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