Introduction: Silk fibroin contains highly repetitive crystalline regions and amorphous regions and has been used as surgical sutures for long time. In the tissue engineering field, it is thought that silk fibroin has potentials to be useful biomaterials due to their good biocompatibility and strength. The secondary structural changes from silk I to silk II of silk fibroin molecules are known to be induced by various stimulations, such as alcohol treatments, heating treatments, pH value and metal ion concentrations. In our previous study, evaluations using 13C solid-state nuclear magnetic resonance (NMR) measurements revealed that structural changes of silk fibroin molecules were induced by autoclaving treatments[1]. Stimulation-induced structural changes of silk fibroin molecules are thought to contribute the immobilization efficiency of colocalized peptides to silk fibroin materials. In this study, our approach to develop functional silk fibroin-based biomaterials is the immobilization of functional peptides to silk fibroin molecules without impairing their excellent properties. Functional peptides[2] containing GAGAGS sequence, one of repetitive sequence in crystalline regions of silk fibroin molecules, were synthesized and immobilized to crystalline regions of silk fibroin-based biomaterials. Analysis of structural changes and immobilization efficiencies of peptides were performed.
Materials and Methods: Silk fibroin aqueous solutions were obtained by conventional methods. Silk fibroin films were prepared by casting methods using silk fibroin aqueous solutions on silicon substrates. Treatments of silk fibroin films with heating or/and alcohol exposing were carried out to induce structural changes of silk fibroin molecules[3]. Influences of these treatments on structural changes of silk fibroin molecules in films were evaluated by 13C solid-state NMR and FTIR spectroscopy. Fluorescence molecules-labelled peptides with/without GAGAGS sequences were synthesized using standard Fmoc-solid state peptide synthesis protocols and purified by HPLC. Immobilization efficiencies of peptides to silk fibroin films were evaluated by the fluorescence microscopy.
Results and Discussion: Surface and bulk structure analysis revealed that β-sheet contents in silk fibroin films were controllable using combination treatments of heating and alcohol exposing. Treatments under 80 % methanol to induce higher β-sheet of silk fibroin films and peptides with GAGAGS sequences yielded specific immobilizations, while treatments using peptides without GAGAGS sequences revealed nonspecific immobilizations. It was reported that RGD peptide containing oligo (D-lactic acid) segments were immobilized to PLLA substrates via stereocomplex formation during preparation of films and fibers[4]. It was surmised that alcohol treatments for silk fibroin films enhanced the immobilization efficiency of colocalized functional peptides containing GAGAGS sequences to silk fibroin materials via β-sheet formations.
Conclusion: Heating or/and alcohol exposing treatments for surfaces of silk fibroin materials are known as general insolubilization methods. Immobilization processing of functional peptides to silk fibroin materials using well-known general treatments would be useful to develop novel functional silk fibroin biomaterials without reductions in mechanical properties or unexpected structural changes of silk fibroins.
This work was partly supported by JSPS KAKENHI Grant Number 15K12543.
References:
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