Regenerated Protein Fibers

Keywords

Silk • Fibroin • Dissolution • Silk regeneration • Primary structure • Secondary structure • Drawing • Tensile properties • Artificial biospinning

Bombyx mori silk fibroin was regenerated into fibers, and the structural differences between the native and regenerated fibers were investigated [98Tra]. To produce fibers, degummed natural silk fibers were dissolved (17 %) in 9.3 M LiBr and dialyzed for 72 h. The aqueous fibroin solution obtained was cast into films. Later, the films were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and spun into fibers using a methanol coagulation bath. During the coagulation of the fibers in methanol, the predominant a-helix form found in fibroin converts to the insoluble crystalline p-sheet. Fibers obtained after drawing had an average diameter of 88 pm and were composed of 56 % p-sheet, 13 % a-helix, 23 % p-turn, and 11 % undefined component similar to that seen in natural silk [98Tra]. Table 48.1 provides a comparison of the secondary structure in the natural and drawn and undrawn regenerated fibers.

A microfabricated wet spinning apparatus was developed to produce regenerated silk fibers using low quantities (10 mg) of silk proteins [99Jel]. In this approach, silk was dissolved in a good solvent such as hexafluoroisopropanol and extruded into a bad solvent such as methanol. Molecular alignment and p-sheet formation occur as the fibers are extruded through the aperture and the fibers are further drawn to improve the properties. Impressively, fibers produced using B. mori proteins had tensile properties similar to that of the native fibers. In another study, silk fibroin from B. mori was dissolved (13 %) in N-methylmorpholine-N-oxide (NMMO) and regenerated into fibers, and the structure and properties of the fibers were studied [05Mar]. Fibroin solution was extruded through spinnerets with 100, 200, or 300 pm orifice at an extrusion rate of 4 m/min into an ethanol coagulation bath and was later drawn in air. Morphologically, the diameter of the fibers was dependent on the draw ratio, and the finest fibers obtained had a diameter of

Table 48.1 Secondary structural compositions of natural fibroin and regenerated fibroin fibers [98Tra] B. mori fiber Orientation a-Helix (%) Disordered helix (%) P-Sheet (%) P-Tum (%) Undefined (%) Natural fibroin X 8.4 10.2 48.6 20.9 10.2 У 5.5 7.0 58.1 23.6 11.1 Pseudoisotropic 5.6 7.0 56.5 23.1 10.9 Regenerated fibroin X 5.7 7.1 56.4 23.2 10.9 Undrawn Y 6.0 7.3 56.0 23.2 11.0 Pseudoisotropic 5.9 7.3 56.2 23.2 11.0 Regenerated fibroin X 6.5 8.3 53.3 21.7 10.4 3.5 x drawn Y 1.4 3.6 64.2 23.0 10.5 Pseudoisotropic 2.9 4.8 60.7 22.8 10.5

19 pm. Undrawn fibers showed ridges on the surface due to protein aggregation but disappeared in the drawn fibers as seen in Fig. 48.1. Fibers were predominantly of the fibroin II (P-sheet) structure and had good thermal stability. The p-sheet formation is shown to occur during coagulation due to the realignment of the intra — and intermolecular forces and leads to substantial improvement in properties [03Li]. Concentration of the protein solution and temperature determine the extent of a — and p-sheets and the crystallization in the fibers [00Mag]. Random coil conformation was obtained when dilute silk fibroin solutions were dried between 0 and 50 °C, whereas a — and p-crystals were obtained by casting concentrated

fibroin solutions [00Mag]. From differential scanning calorimetry (DSC) data, it was found that the exothermic peaks at 65 °C were caused due to the formation of P-sheets in Antheraea pernyi silks. The tensile properties were also dependent on the draw ratio, and some of the properties of the fibers are listed in Table 48.2. As seen from the table, the best regenerated fibers obtained have considerably lower tensile properties compared to native silk fibers. Although most regenerated protein fibers from silk fibroin have been produced using ionic solvents, it has been shown that the fibroin directly extracted from silk glands can be dissolved in 1 % (w/w) sodium dodecyl sulfate and used to develop fibers, films, and other protein-based biomaterials [08Man].

Similar to developing regenerated fibers using B. mori silk, the wild silk pro­duced by A. perni silkworm was regenerated using calcium nitrate solution, and the properties of the regenerated fibroin were studied [01Kwe]. Concentration of calcium nitrate and temperature were found to influence the protein dissolution to a large extent. Solubility increased from 0 to 100 % as the concentration was increased from 4 to 7 M. Similarly, 100 % dissolution was achieved when the temperature was between 100 and 130 °C after 3 h with a calcium nitrate concen­tration of 7 M. Dissolution was suggested to change the conformation of the proteins to а-form and random coil form compared to the predominant p-sheets found in the natural silk. However, other studies have shown that the extent of a — or P-sheet formation in regenerated A. perni fibroin can be controlled during coagula­tion [03Li]. In another study, A. perni silk fibroin was dissolved in lithium thiocya­nate and wet spun into films [09Zuo]. Regenerated protein fibers had a diameter of 0.369 mm with irregular cross section. Proteins were mainly in the p-sheet config­uration and showed typical diffraction peaks, but a-helices and random coils were also present. Tensile properties of the fibers obtained were not reported [09Zuo].

Although strictly not a process of regeneration, an artificial method of biospinning silk fiber matrices was adopted, and the fibers and matrices were used as substrates for tissue engineering. Wild silkworms (A. mylitta) in their fifth instar were collected, and fibers were manually (forcefully) drawn from the silk­worm onto glass slides as shown in Fig. 48.2. Fibers obtained were aligned in

Steps in artificial biospinning of regenerated protein fiber matrices from A. mittrei silk

various fashions to develop matrices for tissue engineering. Alternatively, the silkworms were allowed to naturally spin silk onto Teflon-coated glass plates, and the matrices formed (Fig. 48.2) were collected. Fibers and matrices were degummed and later characterized for their properties, and the potential of using the fibers as substrates for tissue engineering was studied [10Man]. Fibers obtained by forceful extrusion and drawing were circular and had diameters of 12-15 ^m compared to 30-35 ^m for naturally extruded silk. Similarly, the biospun fibers had a tensile strength of 4.1 ± 1.4 g/den, similar to that of B. mori and much higher than that of the natural fibers from A. mylitta. The fibers and matrices developed had enhanced stability to degradation by proteases and found to have good compatibil­ity and supported the attachment and proliferation of fibroblasts [10Man].

References

[98Tra] Trabbic, K. A., Yager, P.: Macromolecules 31, 462 (1998)

[99Jel] Jelinski, L. W., Blye, A., Liivak, O., Michal, C., Verde, G., Seidel, A., Shah, N., Yang, Z.: Int. J. Biol. Macromol. 24, 197 (1999)

[00Mag] Magoshi, J., Magoshi, Y., Becker, M. A., Kato, M., Han, Z., Tanaka, T., Inoue, S., Nakamura, T.: Thermochim. Acta 352-353, 165 (2000)

[01Kwe] Kweon, H., Park, Y. H.: J. Appl. Polym. Sci. 82, 750 (2001)

[03Li] Li, M., Tao, W., Kuga, S., Nishiyama, Y.: Polym. Adv. Technol. 14, 694 (2003)

[05Mar] Marsano, E., Corsini, P., Arosio, C., Boschi, A., Mormino, M., Freddi, G.: Int. J. Biol.

Macromol. 37, 179 (2005)

[08Man] Mandal, B. B., Kundu, S. C.: Biotechnol. Bioeng. 100(6), 1237 (2008)

[09Zuo] Zuo, B., Liu, L., Zhang, F.: J. Appl. Polym. Sci. 113, 2160 (2009)

[10Man] Mandal, B. B., Kundu, S. C.: Acta Biomater. 6(2), 360 (2010)