Effects of phosphate, Tris, HEPES, or MOPS buffers on the formation of silk fibroin sponges

抄録

We investigated the effects of the addition of buffers (pH 7.4 at 25°C) to a silk fibroin (SF) aqueous solution containing 1% (v/v) dimethylsulfoxide on the formation of a SF spongy structure obtained using a single freeze/thawing of the solution. The addition of a sodium phosphate (a mixture of Na₂HPO₄ and NaH₂PO₄; SP) buffer made the SF spongy structure finer and harder dose-dependently, showing the minimum pore size and maximum tensile modulus at 80 mM. In contrast, tris(hydroxymethyl)aminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 3-(N-morpholino)propanesulfonic acid buffers inhibited the SF sponge formation. We infer that the salting in/out effect of ions in buffers affected the SF sponge formation; HPO₄^2- and H₂PO₄^- from SP might be strongly hydrated to make SF proteins assemble by hydrophobic interaction. Because the buffering effect of SP is reportedly insulated from the influence of temperature, the pH of the SF solution was expected to be maintained as around 7.4 during the freeze–thawing process to form the SF sponge. The addition of the SP buffer to the SF solution was likely to maintain the activity of SF proteins modified with pH-sensitive bioactive proteins, even after fabrication into the spongy structure. Furthermore, the resulting SF sponge might show high mechanical properties. Because recent advances in transgenic silkworm technology have enabled the production of recombinant SF protein fused to bioactive proteins, the findings in this study are applicable in the formation of bioactive SF sponges to be used as scaffolds for tissue engineering.