In vivo bone regeneration ability of different layers of natural silk cocoon processed using an eco-friendly method

Silk cocoons are primarily composed of the proteins fibroin and sericin. To achieve guided bone regeneration (GBR), we have developed a simple and ecofriendly processing technique to obtain microperforated thin membranes from cocoons. The separated silk membranes composed of both fibroin and sericin were classified by the cocoon layers (i.e., inner, middle, or outer) from which they originated. This report details the biological properties and the cellular responses of the three silk layers. The different cocoon layers were compared for their bone regeneration capabilities in vivo. The porosity of the silk nets increased from the inner layer to the outer layer when all of the membranes were compared using scanning electron microscopy (SEM). A difference in spectral intensity was observed in the Fourier transform infrared (FT-IR) spectra, indicating different amino acid compositions in these layers. An amino acid composition test demonstrated that the serine content decreased from the outer layer to the inner layer. Characterization of the protein release from each net demonstrated that the highest amount of protein release was observed in the inner layer group. The middle layer showed higher alkaline phosphatase (ALP) activity than the other layers in cellular experiments. Animal experiments indicated that the middle layers exhibit the highest bone volume 8 weeks post-operation (p < 0.05). The membranes obtained directly from the thin middle layer of silk cocoons without any regeneration protocol have the potential to be used as an eco-friendly bone regeneration material for in vivo applications.