Multiscale design and synthesis of biomimetic gradient protein/biosilica composites for interfacial tissue engineering

Jin Guo,1,2 Chunmei Li,2 Shengjie Ling,2,3 Wenwen Huang,2 Ying Chen,2 David L. Kaplan*,2

1Department of Chemical and Biological Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA2Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA3Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Supplementary material

Materials and Methods

Quantification of silicification. The amount of silica in the composite materials was determined using the silicon test colorimetric assay kit (Merck, NJ, USA) according to the manufacturer’s instructions [1–3]. The GSSR5 composites were evenly cut to three sections along the longitude direction of the sample, as referred to the low, medium and high regions. The sections were then washed three times with ethanol to remove unreacted TEOS and hydrolyzed with 1 M NaOH overnight. The samples were diluted to quantify the released silicic acid.

Immunohistochemistry. Samples were harvested after 6 weeks of culture in osteogenic media and fixed in 4% PBS-buffered paraformaldehyde overnight. The samples were dehydrated through graded ethanol (70%, 80%, 90%, 95% and 100%), embedded in paraffin and sectioned (8 µm). Immunostaining of collagen type I were performed based on standard immumohistochemical techniques. Briefly, tissue sections were rehydrated and treated with 5% BSA for 1 h. The samples were treated with anti-collagen I antibodies (Abcam, UK) for overnight at 4°C and then washed with PBS. The samples were stained with secondary antibodies (Abcam, UK) for 1 h at room temperature and washed with PBS. Images were taken on a BZX-700 microscope (Keyence Corporation, IL).

Characterization of hydration properties. The lyophilized GSSR5 composites were evenly cut to three sections along the longitude direction of the composites and tested separately as the low, medium and high regions. Samples were immersed in distilled water at room temperature for 24 h followed by removal of excess water. The wet weight of the hydrated sample (Wh) were determined individually. Samples were then dried in an oven at 60°C for 24 h and the dry weight of samples (Wd) was determined. The swelling ratios and the water uptake percentage were calculated as follows [4]:

Swelling ratio=W h−W d

W d

Water uptake (% )=W h−W d

W h×100

Cell culture and histology. HMSCs were seeded into the GSSR5 composites based on procedures described earlier. The cultures were maintained in chondrogenic differentiation medium at 37°C supplemented with 5% CO2. The medium was changed every 2-3 days. The proteoglycan

* Corresponding author: D.L. Kaplan; E-mail address: david.kaplan@tufts.edu

expression of hMSCs in three-dimensional (3D) cell culture was visualized by Alcian blue staining following the manufacturer’s instructions.

Results

Figure S1. Optical (A) image of the GSSR5 composites. Fluorescence image (B) of the FITC-GSSR5 composites. Fluorescence intensity along the gradient trend of FITC-R5 in the GSSR5 composites (C). Si distribution along the gradient composites generated by EDS mapping (D). Scale bar: 1 mm (A, B), 10 µm (D).

Figure S2. Characterization of pore sizes in the gradient composites. (Significantly difference: *, p < 0.05; **, p<0.01)

Figure S3. Characterization of silica density in the gradient composites. (*Significantly difference (p < 0.05))

Figure S4. Extent of mineralization (measured as the molar percentage of silicon) in the GSSR5 composites with different loadings using silicon test colorimetric assay. Increased silicon content from the low to the high region in the GSSR5 composites demonstrated the gradient mineralization in the composites.

Figure S5. Characterization of the area ratio of calcium staining by von Kossa in the gradient composites. (**Significantly difference (p < 0.01))

Figure S6. Immunostaining of collagen type I taken from the unsilicified plain silk controls (A), and the GSSR5 composites with low (B), medium (C) and high (D) loadings of the R5 peptide after 6-week culture of hMSCs in osteogenic medium. Scale bar, 50 μm.

Figure S7. Characterization of swelling ratio in the gradient composites.

Figure S8. Characterization of water uptake percentage in the gradient composites.

Figure S9. Alcian blued staining of histological sections taken from the GSSR5 composites with low (A), medium (B) and high (C) loadings of the R5 peptide after 4-week culture of hMSCs in chondrogenic medium. Scale bar, 50 μm.

Figure S10. Characterization of the area ratio of collagen staining by Sirius red in the gradient composites.

References:

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