Modification of silk fibroin using diazonium coupling chemistry and the effects on hMSC proliferation and differentiation

Abstract

A simple chemical modification method using diazonium coupling chemistry was developed to tailor the structure and hydrophilicity of silk fibroin protein. The extent of modification using several aniline derivatives was characterized using UV-vis and 1H NMR spectroscopies, and the resulting protein structure was analyzed with ATR-FTIR spectroscopy. Introduction of hydrophobic functional groups facilitated rapid conversion of the protein from a random coil to a beta-sheet structure, while addition of hydrophilic groups inhibited this process. hMSCs were grown on these modified silks to assess the biocompatibility of these materials. The hydrophilicity of the silk derivatives was found to affect the growth rate and morphology, but hMSCs were able to attach, proliferate and differentiate into an osteogenic lineage on all of the silk derivatives.

Figure 1

Aniline derivatives used to modify silk.

Figure 2

a) UV/vis spectra (normalized to the carbonyl peak at 210 nm) in water at pH 7, b) aromatic region of the ¹H-NMR spectra and c) estimated number of tyrosine residues modified based on UV absorbance for sulfonic acid azosilks modified with increasing amounts of (4). *Molar equivalent of diazonium salt added relative to the number of tyrosine residues assuming there are ~115 tyrosines per protein. **Estimated percent of tyrosines modified in each silk molecule.

Figure 3

Comparison of the UV/vis spectra of unmodified and modified silks in water at pH 7 (normalized to the carbonyl peak at 210 nm). The silks were each treated with 0.9 equivalents of diazonium salt relative to the number of tyrosine residues. *Estimated percent of tyrosines modified in each silk molecule assuming that there are ~115 tyrosines per protein, and the extinction coefficients of the azo derivatives are comparable.

Figure 4

Comparison of the ATR-FTIR spectra of unmodified and modified silk films. After treatment with 0.9 equivalents of diazonium salt relative to the number of tyrosine residues, purified solutions of the azosilks in water (3 wt %) were cast into films, allowed to sit for one hour then dried. Ketone azosilk-3 and heptyl azosilk-5 spontaneously form hydrogels, and exhibit shifts in the FTIR spectra consistent with β-sheet formation. Amino azosilk-2 can also spontaneously form hydrogels, but only after 1–2 days. Therefore, the βsheet structure is not observed in these rapidly dried films. For reference, the FTIR spectrum of an unmodified silk film treated with methanol is included, as methanol treatment also induces β-sheet formation.

Figure 5

Measure of the metabolic activity using the alamarBlue^® assay for cells in growth medium on the different substrates at day 4, 7, 9 and 12. Each point represents the mean and standard deviation of N=3 independent cultures, and corrected for the background fluorescence of the dye alone. p<0.05 *relative to cells on unmodified silk, ^# relative to cells on carboxylic acid azosilk-1, ^φrelative to cells on amino azosilk-2, ^δrelative to cells on sulfonic acid azosilk-4, ^εrelative to cells on heptyl azosilk-5 (For clarity, statistical significance between the azosilk derivatives is only given for Day 12.)

Figure 6

Phase contrast microscope images of hMSC morphology when grown on the azosilks and unmodified silk after a) five days and b) 12 days in culture.

Figure 7

Phase contrast and LIVE/DEAD fluorescent images of cells grown on carboxylic acid azosilk-1 and heptyl azosilk-5. Very few dead cells (stained red) are seen indicating that clustered cells are still viable.

Figure 8

Comparison of gene expression on day 12 of the integrin subunits α_v, β₃, α₅ and β₁ from cells grown on the different silk derivatives or tissue culture plastic. Values given are relative to the housekeeping gene GAPDH. Each point represents the mean and standard deviation of N=3 independent cultures. p<0.05 *relative to cells on unmodified silk, ^#relative to cells on carboxylic acid azosilk-1, ^φ relative to cells on amino azosilk-2, ^δrelative to cells on sulfonic acid azosilk-4.

Figure 9

Osteogenic gene expression of cells grown on the different silk derivatives or tissue culture plastic after exposure to osteogenic stimulants for 7 days (+OS) as compared to cells in control medium (−OS). a) α-procollagen I and b) alkaline phosphatase gene expression relative to the housekeeping gene GAPDH. Each point represents the mean and standard deviation of N=3 independent cultures. p<0.05 *relative to cells on unmodified silk, ^#relative to cells on carboxylic acid azosilk-1, ^φ relative to cells on amino azosilk-2.

Scheme 1

Diazonium coupling reaction with tyrosine residues in silk.