Effect of different cocoon stifling methods on the properties of silk fibroin biomaterials.

Stifling treatments are applied to silk cocoons in order to kill the pupae, preventing the emergence of moths and allowing to preserve the silk during long periods of time. All of them involve the application of aggressive steps, such as sun exposure, hot steam from boiling water or hot air, during hours or even days. None of the scientific articles related to silk fibroin biomaterials has previously taken into account this fact in its section of materials and methods. In this work, the consequences of the stifling treatments most commonly used by the silk producing countries and companies are explored in depth, using fibroin films as biomaterial model. The protein degradation (visualised by SDS-PAGE) was dramatically increased in all the fibroin dissolutions produced from stifled cocoons; heavy and light chains of fibroin were specially degraded, reducing their presence along the lanes of the gel compared to the negative control (untreated fresh cocoons). Structural changes are also described for annealed silk fibroin films. The β-sheet content, analysed by means of infrared spectroscopy, was significantly higher when stifling was performed at higher temperature (70 °C and 85 °C). It is also exposed the impact of the stifling on the mechanical properties of the materials. Tensile strength and strain at break values were detected as significantly lower when this procedure was carried out by means of dry heat (85 °C) and sun exposure. On the other hand, and contrary to expectations, the proliferation of fibroblasts growing on the materials was improved by all the different stifling methods, compared to negative control, being this improvement, especially accentuated, on the films produced with fibroin purified from cocoons treated with dry heat.