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

Abstract

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.

Revealing the Influence of the Degumming Process in the Properties of Silk Fibroin Nanoparticles

“…When processing the SF into SFN, the overall β-Sheet structure increased while the rest of secondary structures were reduced for all samples, in agreement with similar works [45]. It could be hypothesized that the relative increase in the β-Sheet content may be caused by degradation of the amorphous regions in peptides of low molecular weight.…”

“…Silk cocoons (SC) were obtained from silkworms Bombyx mori reared in the sericulture facilities of IMIDA (Murcia, Spain) and raised on a diet of fresh, natural Morus alba L. leaves. Cocoons were then stifled to kill the pupae by means of dry heat (85 • C) [45]. The intact chrysalides were extracted manually from the cocoons prior to silk processing.…”

“…For most medical applications of silk biomaterials [1], silk fibroin (SF) must be efficiently purified from silk cocoons (SC) by removing the silk sericin (SS) by means of the process known as degumming, and this key step is commonly recognized as one of the most important procedures in the production of silk-based materials [2]. This purified SF can be dissolved in solutions, such as LiBr 9.3 mol/L [3] or Ajisawa's reagent (CaCl 2 /EtOH/H 2 O, 1:2:8 in molar ratio) [4], the most commonly used, but also in some ionic liquids, such as 1-butyl-3-methylimidazolium chloride ([bmim + ][Cl − ]) [5,6] or The present study focuses on how the degumming process affects SFNs structural features (morphology, size distribution, Z-potential, and surface charge density), intending to delve into the effects of all the stages of the silk processing in the properties of the biomaterials, specifically focused on the reproducibility of the processes [6,12,38,45]. In order to assess the different silk degumming processes and their possible effect on SFNs, silk was degummed by means of four different methods, namely autoclaving (D1), alkaline treatment with Na 2 CO 3 for 30 min (D2) or 120 min (D3), and a high-power ultrasound treatment (D4).…”

Revealing the Influence of the Degumming Process in the Properties of Silk Fibroin Nanoparticles

“…Not surprisingly, SF-D1 and SF-D3 presented the highest CI, both 59 %, but, in our opinion, due to different factors. On the one hand, the alkaline carbonate treatment has the potential to partially degrade the SF fibers, especially those regions with lower crystallinity, resulting in an increase in the overall CI%, an effect previously reported in literature [45]. This result is in accordance with the highest mass loss among samples (44.4%) observed during the treatment.…”

“…Following a priority research line of our lab that attempts to delve into the effects of all the stages of silk processing in the properties of the biomaterials, specifically focused on the reproducibility of the processes, [13,[42][43][44], the present study focuses on how the degumming process affects SFNs structural features (morphology, size distribution, Z-potential and surface charge density). In order to assess the different silk degumming processes and their possible effect on SFNs, silk was degummed by means of four different methods, namely autoclaving (D1), alkaline treatment with Na2CO3 for 30 minutes (D2) or 120 minutes (D3) and a high-power ultrasound treatment (D4).…”