Thermal annealed silk fibroin membranes for periodontal guided tissue regeneration

  • Catarina Geão
  • Ana R. Costa-Pinto
  • Cassilda Cunha-Reis
  • Viviana P. Ribeiro
  • Sílvia Vieira
  • Joaquim M. Oliveira
  • Rui L. Reis
  • Ana L. OliveiraEmail author
Tissue Engineering Constructs and Cell Substrates Original Research
Part of the following topical collections:
  1. Tissue Engineering Constructs and Cell Substrates


Guided tissue regeneration (GTR) is a surgical procedure applied in the reconstruction of periodontal defects, where an occlusive membrane is used to prevent the fast-growing connective tissue from migrating into the defect. In this work, silk fibroin (SF) membranes were developed for periodontal guided tissue regeneration. Solutions of SF with glycerol (GLY) or polyvinyl alcohol (PVA) where prepared at several weight ratios up to 30%, followed by solvent casting and thermal annealing at 85 °C for periods of 6 and 12 h to produce high flexible and stable membranes. These were characterized in terms of their morphology, physical integrity, chemical structure, mechanical and thermal properties, swelling capability and in vitro degradation behavior. The developed blended membranes exhibited high ductility, which is particular relevant considering the need for physical handling and adaptability to the defect. Moreover, the membranes were cultured with human periodontal ligament fibroblast cells (hPDLs) up to 7 days. Also, the higher hydrophilicity and consequent in vitro proteolytic degradability of these blends was superior to pure silk fibroin membranes. In particular SF/GLY blends demonstrated to support high cell adhesion and viability with an adequate hPDLs’ morphology, make them excellent candidates for applications in periodontal regeneration.



The authors acknowledge Portuguese National Funds from FCT - Fundação para a Ciência e a Tecnologia through project UID/Multi/50016/2013; Program FCT Investigators to A.L.Oliveira (IF/00411/2013) and J.M.Oliveira (IF/00423/2012 and IF/01285/2015); PhD scholarship under the financial support from FCT/MCTES and FSE/POCH, PD/59/2013 attributed to V.P.Ribeiro (PD/BD/113806/2015); Project SERICAMED (IF/00411/2013/CP1167); Project “IBEROS” (0245_IBEROS_1_E), funded by Fundo Europeu de Desenvolvimento Regional in the frame of Programa Interreg V A Espanha - Portugal (POCTEP) 2014–2020. This article is a result of the project “Biotherapies” (NORTE-01-0145-FEDER-000012) supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Polimeni G, Xiropaidis AV, Wikesjö UME. Biology and principles of periodontal wound healing/regeneration. Periodontol 2000. 2006;41:30–47.CrossRefGoogle Scholar
  2. 2.
    Nanci A, Bosshardt DD. Periodontal tissues in health and disease. Periodontol 2000. 2006;40:11–28.CrossRefGoogle Scholar
  3. 3.
    Nanci A, Bosshardt DD. Structure of periodontal tissues in health and disease. Periodontol 2000. Periodontol 2000. 2006;40:11–28.CrossRefGoogle Scholar
  4. 4.
    Tariq M, Iqbal Z, Ali J, Baboota S, Talegaonkar S, Ahmad Z, et al. Treatment modalities and evaluation models for periodontitis. Int J Pharm Investig. 2012;2:106–22.CrossRefGoogle Scholar
  5. 5.
    Eke PI, Dye BA, Wei L, Slade GD, Thornton-Evans GO, Borgnakke WS, et al. Update on prevalence of periodontitis in adults in the United States: NHANES 2009 to 2012. J Periodontol. 2015;86:611–22.CrossRefGoogle Scholar
  6. 6.
    Deas DE, Moritz AJ, Sagun RS, Gruwell SF, Powell CA. Scaling and root planing vs. conservative surgery in the treatment of chronic periodontitis. Periodontol 2000. 2016;71:128–39.CrossRefGoogle Scholar
  7. 7.
    Sbordone L, Ramaglia L, Gulletta E, Iacono V. Recolonization of the subgingival microflora after scaling and root planing in human periodontitis. J Periodontol. 1990;61:579–84.CrossRefGoogle Scholar
  8. 8.
    Crea A, Deli G, Littarru C, Lajolo C, Orgeas GV, Tatakis DN. Intrabony defects, open-flap debridement, and decortication: a randomized clinical trial. J Periodontol. 2014;85:34–42.CrossRefGoogle Scholar
  9. 9.
    Mitani A, Takasu H, Horibe T, Furuta H, Nagasaka T, Aino M, et al. Five‐year clinical results for treatment of intrabony defects with EMD, guided tissue regeneration and open‐flap debridement: a case series. J Periodontal Res. 2015;50:123–30.CrossRefGoogle Scholar
  10. 10.
    Hanes PJ. Bone replacement grafts for the treatment of periodontal intrabony defects. Oral Maxillofac Surg Clin North Am Elsevier. 2007;19:499–512.CrossRefGoogle Scholar
  11. 11.
    Mathur A, Bains VK, Gupta V, Jhingran R, Singh GP. Evaluation of intrabony defects treated with platelet-rich fibrin or autogenous bone graft: a comparative analysis. Eur J Dent. 2015;9:100.CrossRefGoogle Scholar
  12. 12.
    Campana V, Milano G, Pagano E, Barba M, Cicione C, Salonna G, et al. Bone substitutes in orthopaedic surgery: from basic science to clinical practice. J Mater Sci Mater Med. 2014;25:2445–61.CrossRefGoogle Scholar
  13. 13.
    Hämmerle CHF, Jung RE. Bone augmentation by means of barrier membranes. Periodontol 2000. 2003;33:36–53.CrossRefGoogle Scholar
  14. 14.
    Gamal AY, Iacono VJ. Enhancing guided tissue regeneration of periodontal defects by using a novel perforated barrier membrane. J Periodontol. 2013;84:905–13.CrossRefGoogle Scholar
  15. 15.
    Al Machot E, Hoffmann T, Lorenz K, Khalili I, Noack B. Clinical outcomes after treatment of periodontal intrabony defects with nanocrystalline hydroxyapatite (Ostim) or enamel matrix derivatives (Emdogain): a randomized controlled clinical trial. Biomed Res Int. 2014;786353:1–9.Google Scholar
  16. 16.
    Hoffmann T, Al-Machot E, Meyle J, Jervøe-Storm P-M, Jepsen S. Three-year results following regenerative periodontal surgery of advanced intrabony defects with enamel matrix derivative alone or combined with a synthetic bone graft. Clin Oral Investig. 2016;20:357–64.CrossRefGoogle Scholar
  17. 17.
    Anitha CM, Senthilkumar S, Rajasekar S, Arun RT, Srinivasan S. Platelet rich fibrin and nanocrystalline hydroxyapatite: hope for regeneration in aggressive periodontitis: a novel clinical approach. Int J Appl Dent Sci. 2017;3:209–14.Google Scholar
  18. 18.
    Ajwani H, Shetty S, Gopalakrishnan D, Kathariya R, Kulloli A, Dolas RS, et al. Comparative evaluation of platelet-rich fibrin biomaterial and open flap debridement in the treatment of two and three wall intrabony defects. J Int Oral Health. 2015;7:32.Google Scholar
  19. 19.
    Sam G, Madhavan Pillai BR. Evolution of barrier membranes in periodontal regeneration—“are the third generation membranes really here?”. J Clin Diagn Res. 2014;8:ZE14–7.Google Scholar
  20. 20.
    Bottino MC, Thomas V, Schmidt G, Vohra YK, Chu TMG, Kowolik MJ, et al. Recent advances in the development of GTR/GBR membranes for periodontal regeneration—a materials perspective. Dent Mater. 2012;28:703–21.CrossRefGoogle Scholar
  21. 21.
    Karring T, Nyman S, Gottlow J, Laurel L. Development of the biological concept of guided tissue regeneration-animal and human studies. Periodontol 2000. 1993;1:26–35.CrossRefGoogle Scholar
  22. 22.
    Llambés F, Silvestre F-J, Caffesse R. Vertical guided bone regeneration with bioabsorbable barriers. J Periodontol. 2007;78:2036–42.CrossRefGoogle Scholar
  23. 23.
    Scantlebury TV. 1982–1992: a decade of technology development for guided tissue regeneration. J Periodontol. 1993;64:1129–37.CrossRefGoogle Scholar
  24. 24.
    Simian M, Dahlin C, Blair K, Schenk RK. Effect of different microstructures of e‐PTFE membranes on bone regeneration and soft tissue response: a histologic study in canine mandible. Clin Oral Implants Res. 1999;10:73–84.CrossRefGoogle Scholar
  25. 25.
    Donos N, Kostopoulos L, Karring T. Alveolar ridge augmentation using a resorbable copolymer membrane and autogenous bone grafts. Clin Oral Implants Res. 2002;13:203–13.CrossRefGoogle Scholar
  26. 26.
    Rothamel D, Schwarz F, Sager M, Herten M, Sculean A, Becker J. Biodegradation of differently cross‐linked collagen membranes: an experimental study in the rat. Clin Oral Implants Res. 2005;16:369–78.CrossRefGoogle Scholar
  27. 27.
    Bunyaratavej P, Wang H-L. Collagen membranes: a review. J Periodontol. 2001;72:215–29.CrossRefGoogle Scholar
  28. 28.
    Zhang JG, Mo XM. Current research on electrospinning of silk fibroin and its blends with natural and synthetic biodegradable polymers. Front Mater Sci. 2013;7:129–42.CrossRefGoogle Scholar
  29. 29.
    Sheikh Z, Qureshi J, Alshahrani AM, Nassar H, Ikeda Y, Glogauer M, et al. Collagen based barrier membranes for periodontal guided bone regeneration applications. Odontology. 2017;105:1–12.CrossRefGoogle Scholar
  30. 30.
    Ueyama Y, Ishikawa K, Mano T, Koyama T, Nagatsuka H, Suzuki K, et al. Usefulness as guided bone regeneration membrane of the alginate membrane. Biomaterials. 2002;23:2027–33.CrossRefGoogle Scholar
  31. 31.
    Ishikawa K, Ueyama Y, Mano T, Koyama T, Suzuki K, Matsumura T. Self‐setting barrier membrane for guided tissue regeneration method: initial evaluation of alginate membrane made with sodium alginate and calcium chloride aqueous solutions. J Biomed Mater Res. 1999;47:111–5.CrossRefGoogle Scholar
  32. 32.
    Xu C, Lei C, Meng L, Wang C, Song Y. Chitosan as a barrier membrane material in periodontal tissue regeneration. J Biomed Mater Res B Appl Biomater. 2012.1435–43.Google Scholar
  33. 33.
    Zhang S, Huang Y, Yang X, Mei F, Ma Q, Chen G, et al. Gelatin nanofibrous membrane fabricated by electrospinning of aqueous gelatin solution for guided tissue regeneration. J Biomed Mater Res A. 2009;90:671–9.CrossRefGoogle Scholar
  34. 34.
    Wang J, Wang L, Zhou Z, Lai H, Xu P, Liao L, et al. Biodegradable polymer membranes applied in guided bone/tissue regeneration: a review. Polym (Basel). 2016;8:115.CrossRefGoogle Scholar
  35. 35.
    Vepari C, Kaplan DL. Silk as a biomaterial. Prog Polym Sci. 2007;32:991–1007.Google Scholar
  36. 36.
    Kim J-Y, Yang B-E, Ahn J-H, Park SO, Shim H-W. Comparable efficacy of silk fibroin with the collagen membranes for guided bone regeneration in rat calvarial defects. J Adv Prosthodont. 2014;6:539–46.CrossRefGoogle Scholar
  37. 37.
    Zhang C, Song D, Lu Q, Hu X, Kaplan DL, Zhu H. Flexibility regeneration of silk fibroin in vitro. Biomacromolecules. 2012;13:2148–53.CrossRefGoogle Scholar
  38. 38.
    Silva MF, De Moraes MA, Nogueira GM, Rodas ACD, Higa OZ, Beppu MM. Glycerin and ethanol as additives on silk fibroin films: Insoluble and malleable films. J Appl Polym Sci. 2013;128:115–22.CrossRefGoogle Scholar
  39. 39.
    Lu Q, Hu X, Wang X, Kluge JA, Lu S, Cebe P, et al. Water-insoluble silk films with silk I structure. Acta Biomater. 2010;6:1380–7.CrossRefGoogle Scholar
  40. 40.
    Motta A, Fambri L, Migliaresi C. Regenerated silk fibroin films: thermal and dynamic mechanical analysis. Macromol Chem Phys. 2002;203:1658–65.CrossRefGoogle Scholar
  41. 41.
    Correia C, Bhumiratana S, Yan L-P, Oliveira AL, Gimble JM, Rockwood D, et al. Development of silk-based scaffolds for tissue engineering of bone from human adipose-derived stem cells. Acta Biomater. 2012;8:2483–92.CrossRefGoogle Scholar
  42. 42.
    Owens DK, Wendt RC. Estimation of the surface free energy of polymers. J Appl Polym Sci. 1969;13:1741–7.CrossRefGoogle Scholar
  43. 43.
    Kaelble DH. Dispersion-polar surface tension properties of organic solids. J Adhes. 1970;2:66–81.CrossRefGoogle Scholar
  44. 44.
    Horan RL, Antle K, Collette AL, Wang Y, Huang J, Moreau JE, et al. In vitro degradation of silk fibroin. Biomaterials. 2005;26:3385–93.CrossRefGoogle Scholar
  45. 45.
    Almeida LR, Martins AR, Fernandes EM, Oliveira MB, Correlo VM, Pashkuleva I, et al. New biotextiles for tissue engineering: development, characterization and in vitro cellular viability. Acta Biomater. 2013;9:8167–81.CrossRefGoogle Scholar
  46. 46.
    Lu S, Wang X, Lu Q, Zhang X, Kluge JA, Uppal N, et al. Insoluble and flexible silk films containing glycerol. Biomacromolecules ACS Publ. 2009;11:143–50.CrossRefGoogle Scholar
  47. 47.
    Tsukada M, Freddi G, Crighton JS. Structure and compatibility of poly (vinyl alcohol)‐silk fibroin (PVA/SA) blend films. J Polym Sci B Polym Phys. 1994;32:243–8.CrossRefGoogle Scholar
  48. 48.
    Tanaka T, Tanigami T, Yamaura K. Phase separation structure in poly (vinyl alcohol)/silk fibroin blend films. Polym Int. 1998;45:175–84.CrossRefGoogle Scholar
  49. 49.
    Hofmann S, CTWP Foo, Rossetti, Textor F, Vunjak-Novakovic M, Kaplan G. DL, et al. Silk fibroin as an organic polymer for controlled drug delivery. J Control Release. 2006;111:219–27.CrossRefGoogle Scholar
  50. 50.
    Ayub ZH, Arai M, Hirabayashi K. Quantitative structural analysis and physical properties of silk fibroin hydrogels. Polym. 1994;35:2197–200.CrossRefGoogle Scholar
  51. 51.
    Chen X, Shao Z, Marinkovic NS, Miller LM, Zhou P, Chance MR. Conformation transition kinetics of regenerated Bombyx mori silk fibroin membrane monitored by time-resolved FTIR spectroscopy. Biophys Chem. 2001;89:25–34.CrossRefGoogle Scholar
  52. 52.
    Indran VP, Zuhaimi NAS, Deraman MA, Maniam GP, Yusoff MM, Hin T-YY, et al. An accelerated route of glycerol carbonate formation from glycerol using waste boiler ash as catalyst. RSC Adv. 2014;4:25257–67.CrossRefGoogle Scholar
  53. 53.
    Sudhamani SR, Prasad MS, Sankar KU.DSC and FTIR studies on gellan and polyvinyl alcohol (PVA) blend films. Food Hydrocoll. 2003;17:245–50.CrossRefGoogle Scholar
  54. 54.
    Dai L, Li J, Yamada E. Effect of glycerin on structure transition of PVA/SF blends. J Appl Polym Sci. 2002;86:2342–7.CrossRefGoogle Scholar
  55. 55.
    Magoshi J, Nakamura S. Studies on physical properties and structure of silk. Glass transition and crystallization of silk fibroin. J Appl Polym Sci. 1975;19:1013–5.CrossRefGoogle Scholar
  56. 56.
    Koosha M, Mirzadeh H, Shokrgozar MA, Farokhi M. Nanoclay-reinforced electrospun chitosan/PVA nanocomposite nanofibers for biomedical applications. RSC Adv. 2015;5:10479–87.CrossRefGoogle Scholar
  57. 57.
    Freddi G, Tsukada M, Beretta S. Structure and physical properties of silk fibroin/polyacrylamide blend films. J Appl Polym Sci. 1999;71:1563–71.CrossRefGoogle Scholar
  58. 58.
    Wang X, Zhang X, Castellot J, Herman I, Iafrati M, Kaplan DL. Controlled release from multilayer silk biomaterial coatings to modulate vascular cell responses. Biomater. 2008;29:894–903.CrossRefGoogle Scholar
  59. 59.
    Sofia S, McCarthy MB, Gronowicz G, Kaplan DL. Functionalized silk‐based biomaterials for bone formation. J Biomed Mater Res. 2001;54:139–48.CrossRefGoogle Scholar
  60. 60.
    Asakura T, Kuzuhara A, Tabeta R, Saito H. 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. Macromoles. 1985;18:1841–5.CrossRefGoogle Scholar
  61. 61.
    Chen X, Li W, Yu T. Conformation transition of silk fibroin induced by blending chitosan. J Polym Sci Phys. 1997;35:2293–6.CrossRefGoogle Scholar
  62. 62.
    Kweon H, Woo SO, Park YH. Effect of heat treatment on the structural and conformational changes of regenerated Antheraea pernyi silk fibroin films. J Appl Polym Sci. 2001;81:2271–6.CrossRefGoogle Scholar
  63. 63.
    Vieira MGA, Da Silva MA, Dos Santos LO, Beppu MM. Natural-based plasticizers and biopolymer films: a review. Eur Polym J. 2011;47:254–63.Google Scholar
  64. 64.
    Fill TS, Carey JP, Toogood RW, Major PW, Experimentally determined mechanical properties of, and models for, the periodontal ligament: critical review of current literature. J Dent Biomech. 2011;312980:1–10.Google Scholar
  65. 65.
    Coïc M, Placet V, Jacquet E, Meyer C. [Mechanical properties of collagen membranes used in guided bone regeneration: a comparative study of three models]. Rev Stomatol Chir Maxillofac. 2009;111:286–90.CrossRefGoogle Scholar
  66. 66.
    Milella E, Barra G, Ramires PA, Leo G, Aversa P, Romito A. Poly (L‐lactide) acid/alginate composite membranes for guided tissue regeneration. J Biomed Mater Res 2001;57:248–57.CrossRefGoogle Scholar
  67. 67.
    Holland C, Numata K, Rnjak-Kovacina J, Seib FP. The biomedical use of silk: past, present, future. Adv Health Care Mater. 2019;8:1–26.Google Scholar
  68. 68.
    Villar CC, Cochran DL. Regeneration of periodontal tissues: guided tissue regeneration. Dent Clin North Am. 2010;54:73–92.Google Scholar
  69. 69.
    Brown J, Lu CL, Coburn J, Kaplan DL. Impact of silk biomaterial structure on proteolysis. Acta Biomater 2015;11:212–21.Google Scholar
  70. 70.
    Yang Y, Zhao Y, Gu Y, Yan X, Liu J, Ding F, et al. Degradation behaviors of nerve guidance conduits made up of silk fibroin in vitro and in vivo. Polym Degrad Stab. 2009;94:2213–20.Google Scholar
  71. 71.
    Zhou J, Cao C, Ma X, Hu L, Chen L, Wang C. In vitro and in vivo degradation behavior of aqueous-derived electrospun silk fibroin scaffolds. Polym Degrad Stab. 2010;95:1679–85.Google Scholar
  72. 72.
    Cai Y, Guo J, Chen C, Yao C, Chung SM, Yao J, et al. Silk fibroin membrane used for guided bone tissue regeneration. Mater Sci Eng C Mater Biol Appl. 2017;70:148–54.Google Scholar
  73. 73.
    Lee JM, Sultan MT, Kim SH, Kumar V, Yeon YK, Lee OJ, et al. Artificial auricular cartilage using silk fibroin and polyvinyl alcohol hydrogel. Int J Mol Sci. 2017;18:1707.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Catarina Geão
    • 1
    • 2
  • Ana R. Costa-Pinto
    • 1
  • Cassilda Cunha-Reis
    • 1
  • Viviana P. Ribeiro
    • 2
    • 3
  • Sílvia Vieira
    • 2
    • 3
  • Joaquim M. Oliveira
    • 2
    • 3
    • 4
  • Rui L. Reis
    • 2
    • 3
    • 4
  • Ana L. Oliveira
    • 1
    Email author
  1. 1.CBQF–Centro de Biotecnologia e Química Fina, Laboratório Associado, Escola Superior de BiotecnologiaUniversidade Católica PortuguesaPortoPortugal
  2. 2.3B’s Research Group, I3Bs–Research Institute on Biomaterials, Biodegradable and BiomimeticsHeadquarters of the European Institute of Excellence on tissue Engineering and Regenerative MedicineBarcoPortugal
  3. 3.ICVS/3B’s–PT Government Associated LaboratoryBraga/GuimarãesPortugal
  4. 4.The Discoveries Centre for Regenerative and Precision MedicineHeadquarters at University of MinhoBarcoPortugal

Personalised recommendations