Silk/polyethylene glycol (PEG) hydrogels are studied as self‐standing bioinks for 3D printing for tissue engineering. The two components of the bioink, silk fibroin protein (silk) and PEG, are both Food and Drug Administration approved materials in drug and medical device products. Mixing PEG with silk induces silk β‐sheet structure formation and thus gelation and water insolubility due to physical crosslinking. A variety of constructs with high resolution, high shape fidelity, and homogeneous gel matrices are printed. When human bone marrow mesenchymal stem cells are premixed with the silk solution prior to printing and the constructs are cultured in this medium, the cell‐loaded constructs maintain their shape over at least 12 weeks. Interestingly, the cells grow faster in the higher silk concentration (10%, w/v) gel than in lower ones (7.5 and 5%, w/v), likely due to the difference in material stiffness and the amount of residual PEG remaining in the gel related to material hydrophobicity. Subcutaneous implantation of 7.5% (w/v) bioink gels with and without printed fibroblast cells in mice reveals that the cells survive and proliferate in the gel matrix for at least 6 week postimplantation. The results suggest that these silk/PEG bioink gels may provide suitable scaffold environments for cell printing and function.
The paper presents a study using silk nano-and microparticles as carriers to deliver curcumin (CUR), a multifunctional and efficient, yet water-insoluble, antioxidant, with the primary goal of enhancing bioavailability. CUR-loaded silk nanoparticles were prepared by emulsification, resulting in particle sizes ranging from 229 to 2286 nm in diameter, with loading efficiencies from 22 to 41%. The encapsulation mechanism involved hydrophobic interaction between the β-sheet structure fabricated from the silk and the phenol groups of curcumin. In vitro studies revealed that curcumin was released more slowly from the larger particles than the smaller particles. An HPLC−MS/MS method was developed to determine the content of curcumin in rat plasma. The particles were orally administered at a single dose in rats, and the pharmacokinetic parameters were evaluated and compared with those of plain curcumin crystals. After 24 h, the larger silk particles (CSN-800, CUR-silk nanoparticles with sizes ∼800 nm) showed longer plasma circulation time for CUR (T max was 1.76 ± 0.10 h) when compared with that of the smaller silk particles (CSN-200 with sizes ∼200 nm, T max was 1.17 ± 0.07 h). The C max of CSN-200 was 3-fold greater than that of CSN-800. By contrast to the groups of plain CUR (without silk particles), CSN-200 and CSN-800 increased CUR bioavailability 17-and 5-fold, respectively. When mixtures of CSN-200 and CSN-800 at different ratios were orally administered to rats, medium bioavailability (>13 times) and exposure time (T 1/2 > 1.68 h) were obtained. These results elucidated the effect of CUR-silk particle sizes on bioavailability after oral delivery, as a basis for future applications.
The stabilities of three natural antioxidants, vitamin C (VC), (-)-epigallocatechin gallate (EGCG), and curcumin, in silk films were examined and mechanisms of stabilization were elucidated. The antioxidants were physically incorporated into three types of silk films: as-cast, dried from hydrogels, and methanol-treated. Films were stored at 4, 37, and 45 °C for 30 days in phosphate-buffered saline, pH 7.4, along with controls consisting of free antioxidants. Incorporation of antioxidants did not significantly change film morphology or secondary structure. When stored at 4 °C, all samples showed similar antioxidant activities (percent scavenging) at different time points, determined by the colorimetric 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. At higher temperatures, VC in the as-cast film, EGCG in the as-cast and dried hydrogel films, and curcumin in the methanol-treated films retained more than 50% scavenging activity after 14 days of storage, significantly higher than the other samples. Interaction between antioxidants and silk, as well as degradation of the antioxidants, was investigated by fast-performance liquid chromatography (FPLC) and high-pressure liquid chromatography (HPLC), with an aim of understanding the mechanisms of silk-based stabilization. Binding of antioxidant molecules to hydrophobic or to hydrophilic/hydrophilic boundary regions of silk, depending on the chemical properties of the antioxidant, may account for the observed stabilization effects. The data can help guide further engineering of antioxidant-functionalized silk biomaterials.
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