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Cell-culture compatible silk fibroin scaffolds concomitantly patterned by freezing conditions and salt concentration

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Abstract

The morphology of freeze-dried silk fibroin 3D-scaffolds was modified by varying both the NaCl concentration and the freezing temperature of the silk fibroin solution prior to lyophilization. Scanning electron micrographs showed that slow freezing at −22 °C generated sponge-like interconnected porous networks, whereas fast freezing at −73 °C formed stacked leaflet structures. The presence of millimolar NaCl (50–250 mM) increased the porosity of the scaffolds and generated small outgrowths at their surface, depending on the freezing regime. Our results suggest that the morphological differences seen between the materials likely depend on ice and NaCl hydrate crystal nucleation and growth mechanisms. Infrared spectroscopy and X-ray diffraction analyses revealed that the salt concentration and freezing conditions induced no structural changes in fibroin. The seeding of P19 embryonic carcinoma cells showed that the presence of salt and freezing conditions influenced the cell distribution into the scaffolds, with salt addition increasing the access of cells to deeper regions.

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References

  1. Kim U-J, Park J, Joo Kim H, Wada M, Kaplan DL (2005) Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin. Biomaterials 26(15):2775–2785

    Article  CAS  Google Scholar 

  2. Agrawal CM, Ray RB (2001) Biodegradable polymeric scaffolds for musculoskeletal tissue engineering. J Biomed Mater Res 55(2):141–150

    Article  CAS  Google Scholar 

  3. Zmora S, Glicklis R, Cohen S (2002) Tailoring the pore architecture in 3-D alginate scaffolds by controlling the freezing regime during fabrication. Biomaterials 23(20):4087–4094

    Article  CAS  Google Scholar 

  4. Hollister SJ (2005) Porous scaffold design for tissue engineering. Nat Mater 4(7):518–524

    Article  CAS  Google Scholar 

  5. Hofmann S, Hagenmüller H, Koch AM, Müller R, Vunjak-Novakovic G, Kaplan DL, Merkle HP, Meinel L (2007) Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials 28(6):1152–1162

    Article  CAS  Google Scholar 

  6. Nazarov R, Jin H-J, Kaplan DL (2004) Porous 3-D scaffolds from regenerated silk fibroin. Biomacromolecules 5(3):718–726

    Article  CAS  Google Scholar 

  7. Sofia S, McCarthy MB, Gronowicz G, Kaplan DL (2001) Functionalized silk-based biomaterials for bone formation. J Biomed Mater Res 54(1):139–148

    Article  CAS  Google Scholar 

  8. Budyanto L, Goh Y, Ooi C (2009) Fabrication of porous poly(l-lactide) (plla) scaffolds for tissue engineering using liquid–liquid phase separation and freeze extraction. J Mater Sci Mater Med 20(1):105–111

    Article  CAS  Google Scholar 

  9. Madihally SV, Matthew HWT (1999) Porous chitosan scaffolds for tissue engineering. Biomaterials 20(12):1133–1142

    Article  CAS  Google Scholar 

  10. Liu CZ, Xia ZD, Han ZW, Hulley PA, Triffitt JT, Czernuszka JT (2008) Novel 3D collagen scaffolds fabricated by indirect printing technique for tissue engineering. J Biomed Mater Res Part B 85B(2):519–528

    Article  CAS  Google Scholar 

  11. Hu Y, Grainger DW, Winn SR, Hollinger JO (2002) Fabrication of poly(alpha-hydroxy acid) foam scaffolds using multiple solvent systems. J Biomed Mater Res 59(3):563–572

    Article  CAS  Google Scholar 

  12. Stokols S, Tuszynski MH (2004) The fabrication and characterization of linearly oriented nerve guidance scaffolds for spinal cord injury. Biomaterials 25(27):5839–5846

    Article  CAS  Google Scholar 

  13. Lee KY, Mooney DJ (2001) Hydrogels for tissue engineering. Chem Rev 101(7):1869–1880

    Article  CAS  Google Scholar 

  14. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, Lu H, Richmond J, Kaplan DL (2003) Silk-based biomaterials. Biomaterials 24(3):401–416

    Article  CAS  Google Scholar 

  15. Vepari C, Kaplan DL (2007) Silk as a biomaterial. Prog Polym Sci 32(8–9):991–1007

    Article  CAS  Google Scholar 

  16. Mandal BB, Kundu SC (2009) Cell proliferation and migration in silk fibroin 3D scaffolds. Biomaterials 30(15):2956–2965

    Article  CAS  Google Scholar 

  17. Perasso R, Zhou CZ, Li ZG, Confalonieri F, Medina N, Zivanovic Y, Esnault C, Yang T, Jacquet M, Janin J, Duguet M (2000) Fine organization of bombyx mori fibroin heavy chain gene. Nucleic Acids Res 28:2413–2419

    Article  Google Scholar 

  18. Inoue S, Tanaka K, Arisaka F, Kimura S, Ohtomo K, Mizuno S (2000) Silk fibroin of bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of h-chain, l-chain, and p25, with a 6:6:1 molar ratio. J Biol Chem 275(51):40517–40528

    Article  CAS  Google Scholar 

  19. Shen Y, Johnson MA, Martin DC (1998) Microstructural characterization of bombyx mori silk fibers. Macromolecules 31(25):8857–8864

    Article  CAS  Google Scholar 

  20. Servoli E, Maniglio D, Motta A, Predazzer R, Migliaresi C (2005) Surface properties of silk fibroin films and their interaction with fibroblasts. Macromol Biosci 5(12):1175–1183

    Article  CAS  Google Scholar 

  21. Horan RL, Antle K, Collette AL, Wang Y, Huang J, Moreau JE, Volloch V, Kaplan DL, Altman GH (2005) In vitro degradation of silk fibroin. Biomaterials 26(17):3385–3393

    Article  CAS  Google Scholar 

  22. Lawrence BD, Cronin-Golomb M, Georgakoudi I, Kaplan DL, Omenetto FG (2008) Bioactive silk protein biomaterial systems for optical devices. Biomacromolecules 9(4):1214–1220

    Article  CAS  Google Scholar 

  23. Lawrence BD, Marchant JK, Pindrus MA, Omenetto FG, Kaplan DL (2009) Silk film biomaterials for cornea tissue engineering. Biomaterials 30(7):1299–1308

    Article  CAS  Google Scholar 

  24. Hino T, Tanimoto M, Shimabayashi S (2003) Change in secondary structure of silk fibroin during preparation of its microspheres by spray-drying and exposure to humid atmosphere. J Colloid Interface Sci 266:68–73

    Article  CAS  Google Scholar 

  25. Zhu J, Shao H, Hu X (2007) Morphology and structure of electrospun mats from regenerated silk fibroin aqueous solutions with adjusting ph. Int J Biol Macromol 41(4):469–474

    Article  CAS  Google Scholar 

  26. Li C, Vepari C, Jin H-J, Kim HJ, Kaplan DL (2006) Electrospun silk-bmp-2 scaffolds for bone tissue engineering. Biomaterials 27(16):3115–3124

    Article  CAS  Google Scholar 

  27. Li M, Wu Z, Zhang C, Lu S, Yan H, Huang D, Ye H (2001) Study on porous silk fibroin materials. II. Preparation and characteristics of spongy porous silk fibroin materials. J Appl Polym Sci 79(12):2192–2199

    Article  Google Scholar 

  28. Uebersax L, Merkle HP, Meinel L (2008) Insulin-like growth factor I releasing silk fibroin scaffolds induce chondrogenic differentiation of human mesenchymal stem cells. J Control Rel 127(1):12–21

    Article  CAS  Google Scholar 

  29. Tamada Y (2005) New process to form a silk fibroin porous 3-D structure. Biomacromolecules 6(6):3100–3106

    Article  CAS  Google Scholar 

  30. Zhang H, Cooper AI (2007) Aligned porous structures by directional freezing. Adv Mater 19(11):1529–1533

    Article  CAS  Google Scholar 

  31. Kwon S-M, Kim H-S, Jin H-J (2009) Multiwalled carbon nanotube cryogels with aligned and non-aligned porous structures. Polymer 50(13):2786–2792

    Article  CAS  Google Scholar 

  32. Ishida M, Asakura T, Yokoi M, Saito H (2002) Solvent- and mechanical-treatment-induced conformational transition of silk fibroins studies by high-resolution solid-state carbon-13 NMR spectroscopy. Macromolecules 23(1):88–94

    Article  Google Scholar 

  33. Bouchard F, Paquin J (2009) Skeletal and cardiac myogenesis accompany adipogenesis in p19 embryonal stem cells. Stem Cells Dev 18(7):1023–1032

    Article  CAS  Google Scholar 

  34. Fukasawa T, Deng ZY, Ando M, Ohji T, Goto Y (2001) Pore structure of porous ceramics synthesized from water-based slurry by freeze-dry process. J Mater Sci 36(10):2523–2527

    Article  CAS  Google Scholar 

  35. Vrbka L, Jungwirth P (2007) Molecular dynamics simulations of freezing of water and salt solutions. J Mol Liq 134(1–3):64–70

    Article  CAS  Google Scholar 

  36. Carignano MA, Baskaran E, Shepson PB, Szleifer I (2006) Molecular dynamics simulation of ice growth from supercooled pure water and from salt solution. Ann Glaciol 44:113–117

    Article  CAS  Google Scholar 

  37. Vrbka L, Jungwirth P (2005) Brine rejection from freezing salt solutions: a molecular dynamics study. Phys Rev Lett 95(14):148501

    Article  Google Scholar 

  38. Hu K, Cui F, Lv Q, Ma J, Feng Q, Xu L, Fan D (2008) Preparation of fibroin/recombinant human-like collagen scaffold to promote fibroblasts compatibility. J Biomed Mater Res Part A 84A(2):483–490

    Article  CAS  Google Scholar 

  39. Zaritzky N (2005) Physical-chemical principles in freezing. In: Sun D-W (ed) Handbook of frozen food processing and packaging. Taylor and Francis group edn. CRC Press, Boca Raton, FL, pp 3–32

    Chapter  Google Scholar 

  40. Nam J, Park YH (2001) Morphology of regenerated silk fibroin: Effects of freezing temperature, alcohol addition, and molecular weight. J Appl Polym Sci 81(12):3008–3021

    Article  CAS  Google Scholar 

  41. Nam YS, Park TG (1999) Biodegradable polymeric microcellular foams by modified thermally induced phase separation method. Biomaterials 20(19):1783–1790

    Article  CAS  Google Scholar 

  42. Marsh RE, Corey RB, Pauling L (1955) An investigation of the structure of silk fibroin. Biochim Biophys Acta 16:1–34

    Article  CAS  Google Scholar 

  43. Asakura T, Kuzuhara A, Tabeta R, Saito H (2002) Conformational characterization of bombyx mori silk fibroin in the solid state by high-frequency carbon-13 cross polarization-magic angle spinning NMR, X-ray diffraction, and infrared spectroscopy. Macromolecules 18(10):1841–1845

    Article  Google Scholar 

  44. He S-J, Valluzzi R, Gido SP (1999) Silk I structure in bombyx mori silk foams. Int J Biol Macromol 24(2–3):187–195

    Article  CAS  Google Scholar 

  45. Lv Q, Cao C, Zhang Y, Ma X, Zhu H (2005) Preparation of insoluble fibroin films without methanol treatment. J Appl Polym Sci 96(6):2168–2173

    Article  CAS  Google Scholar 

  46. Chen X, Shao Z, Marinkovic NS, Miller LM, Zhou P, Chance MR (2001) Conformation transition kinetics of regenerated bombyx mori silk fibroin membrane monitored by time-resolved ftir spectroscopy. Biophys Chem 89(1):25–34

    Article  CAS  Google Scholar 

  47. Sashina E, Bochek A, Novoselov N, Kirichenko D (2006) Structure and solubility of natural silk fibroin. Russ J Appl Chem 79(6):869–876

    Article  CAS  Google Scholar 

  48. Velema J, Kaplan D (2006) Biopolymer-based biomaterials as scaffolds for tissue engineering. In: Lee K, Kaplan D (eds) Tissue engineering I. Advances in biochemical engineering/biotechnology, vol 102. Springer, Berlin, Heidelberg, pp 187–238

    Google Scholar 

  49. Chung T-W, Chang Y-L (2010) Silk fibroin/chitosan–hyaluronic acid versus silk fibroin scaffolds for tissue engineering: promoting cell proliferations in vitro. J Mater Sci Mater Med 21(4):1343–1351

    Article  CAS  Google Scholar 

  50. Skerjanc IS (1999) Cardiac and skeletal muscle development in p19 embryonal carcinoma cells. Trends in Cardiovascul Med 9(5):139–143

    Article  CAS  Google Scholar 

  51. Rudnicki MA, McBurney MW (1987) Cell culture methods and induction of differentiation of embryonal carcinoma cell lines. In: Robertson EJ (ed) Teratocarcinomas and embryonic stem cells: a practical approach. IRL press edn. IRL Press, Oxford, pp 19–49

    Google Scholar 

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Acknowledgments

We are grateful to R. Mineau and D. Flipo for their help with the SEM and scanning confocal microscopy images, respectively. We are also very thankful to D.L. Kaplan and J.A. Kluge (Tufts University) for their kind assistance with the mechanical testing. F.B. thanks Pharmaqam for the award of a scholarship. This work was partly supported by grants from the Natural Sciences and Engineering Research Council of Canada (I.M., C.P. and M.A.M) and the Canadian Institutes for Health Research (J.P.).

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Correspondence to Isabelle Marcotte or Mircea A. Mateescu.

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Byette, F., Bouchard, F., Pellerin, C. et al. Cell-culture compatible silk fibroin scaffolds concomitantly patterned by freezing conditions and salt concentration. Polym. Bull. 67, 159–175 (2011). https://doi.org/10.1007/s00289-010-0438-z

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  • DOI: https://doi.org/10.1007/s00289-010-0438-z

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