Influence of chitosan-chitin nanofiber composites on cytoskeleton structure and the proliferation of rat bone marrow stromal cells

  • Victoria V. Kiroshka
  • Valentina A. Petrova
  • Daniil D. Chernyakov
  • Yulia O. Bozhkova
  • Katerina V. Kiroshka
  • Yulia G. Baklagina
  • Dmitry P. Romanov
  • Roman V. Kremnev
  • Yury A. Skorik
Tissue Engineering Constructs and Cell Substrates Original Research
Part of the following topical collections:
  1. Tissue Engineering Constructs and Cell Substrates


Chitosan scaffolds have gained much attention in various tissue engineering applications, but the effect of their microstructure on cell-material spatial interactions remains unclear. Our objective was to evaluate the effect of chitosan-based matrices doping with chitin nano-whiskers (CNW) on adhesion, spreading, cytoskeleton structure, and proliferation of rat bone marrow stromal cells (BMSCs). The behavior of BMSCs during culture on chitosan-CNW films was determined by the molecular mass, hydrophobicity, porosity, crosslinking degree, protonation degree and molecular structure of the composite chitosan-CNW films. The shape, spreading area, cytoskeleton structure, and proliferation of BMSCs on chitosan matrices with a crystalline structure and high porosity were similar to that observed for BMSCs cultured on polystyrene tissue culture plates. The amorphous polymer structure and high swelling led to a decrease in the spreading area and cell proliferation. Thus, we can control the behavior of cells in culture (adhesion, spreading, and proliferation) by changing the physico-chemical properties of the chitosan-CNW films.


Chitosan Composite Film Shape Index Chitosan Solution Chitosan Film 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



VA Petrova, DD Chernyakov, and YA Skorik are grateful to the Russian Science Foundation (project #16-19-10536) for financial support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Lanza RP, Langer R, Vacanti JP. Principles of tissue engineering. 3rd ed. Academic Press; San Diego, CA, USA. 2011.Google Scholar
  2. 2.
    Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol. 1999;181(1):67–73. doi:10.1002/(SICI)1097-4652(199910)181:1<67::AID-JCP7>3.0.CO;2-C.CrossRefGoogle Scholar
  3. 3.
    Gao J, Yao JQ, Caplan AI. Stem cells for tissue engineering of articular cartilage. Proc Inst Mech Eng H. 2007;221(5):441–50.CrossRefGoogle Scholar
  4. 4.
    Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells. 2001;19(3):180–92. doi: 10.1634/stemcells.19-3-180.CrossRefGoogle Scholar
  5. 5.
    Ciapetti G, Ambrosio L, Marletta G, Baldini N, Giunti A. Human bone marrow stromal cells: in vitro expansion and differentiation for bone engineering. Biomaterials. 2006;27(36):6150–60. doi: 10.1016/j.biomaterials.2006.08.025.CrossRefGoogle Scholar
  6. 6.
    Panarin EF, et al. Matrices for cell culture of human skin cells based on natural polysaccharides chitin and chitosan. Kletochnaya Transplantologiya i Tkanevaya Inzheneriya (Cell Transplantology and Tissue Engineering). 2009;4(3):42–6.Google Scholar
  7. 7.
    Shoichet MS. Polymer scaffolds for biomaterials applications. Macromolecules. 2010;43(2):581–91. doi: 10.1021/ma901530r.CrossRefGoogle Scholar
  8. 8.
    Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials. 2000;21(24):2589–98.CrossRefGoogle Scholar
  9. 9.
    Afanas’eva NV, et al. Molecular mobility of chitosan and its interaction with montmorillonite in composite films: dielectric spectroscopy and FTIR studies. Poly Sci Series A. 2013;55(12):738–48. doi: 10.1134/S0965545X13120018.CrossRefGoogle Scholar
  10. 10.
    Khan A, et al. Mechanical and barrier properties of nanocrystalline cellulose reinforced chitosan based nanocomposite films. Carbohydr Polym. 2012;90(4):1601–8. doi: 10.1016/j.carbpol.2012.07.037.CrossRefGoogle Scholar
  11. 11.
    Petrova VA, et al. Specific features of chitosan-montmorillonite interaction in an aqueous acid solution and properties of related composite films. Poly Sci Series A. 2012;54(3):224–30. doi: 10.1134/S0965545X1203008X.CrossRefGoogle Scholar
  12. 12.
    Shchipunov YA, Silant’ev VE, Postnova IV. Self-organization in the chitosan-clay nanoparticles system regulated through polysaccharide macromolecule charging. 1. Hydrogels. Colloid J. 2012;74(5):627–35. doi: 10.1134/S1061933X12050092.CrossRefGoogle Scholar
  13. 13.
    Fan Y, Saito T, Isogai A. Individual chitin nano-whiskers prepared from partially deacetylated α-chitin by fibril surface cationization. Carbohydr Polym. 2010;79(4):1046–51. doi: 10.1016/j.carbpol.2009.10.044.CrossRefGoogle Scholar
  14. 14.
    Kiroshka VV, et al. Adhesion, growth, and proliferation of endothelial cells on biopolymer extracellular film matrices. Bull Exp Biol Med. 2014;158(1):153–8. doi: 10.1007/s10517-014-2712-9.CrossRefGoogle Scholar
  15. 15.
    Zotkin MA, Vikhoreva GA, Kechek’yan AS. Thermal modification of chitosan films in the form of salts with various acids. Poly Sci Series B. 2004;46(1-2):39–42.Google Scholar
  16. 16.
    Nud’ga LA, et al. Chemical and structural transformations in chitosan films in the course of storage. Russ J Appl Chem. 2008;81(11):1992–6. doi: 10.1134/S1070427208110244.CrossRefGoogle Scholar
  17. 17.
    Pogodina NV, et al. Conformational characteristics of chitosan molecules as demonstrated by diffusion-sedimentation analysis and viscometry. Poly Sci USSR. 1986;28(2):251–9. doi: 10.1016/0032-3950(86)90076-6.CrossRefGoogle Scholar
  18. 18.
    Kim K, Dean D, Mikos AG, Fisher JP. Effect of initial cell seeding density on early osteogenic signal expression of rat bone marrow stromal cells cultured on cross-linked poly(propylene fumarate) disks. Biomacromolecules. 2009;10(7):1810–7. doi: 10.1021/bm900240k.CrossRefGoogle Scholar
  19. 19.
    Anokhina EB, Buravkova LB. Heterogeneity of stromal precursor cells isolated from rat bone marrow. Tsitologiia. 2007;49(1):40–7.Google Scholar
  20. 20.
    Huang Y, Siewe M, Madihally SV. Effect of spatial architecture on cellular colonization. Biotechnol Bioeng. 2006;93(1):64–75. doi: 10.1002/bit.20703.CrossRefGoogle Scholar
  21. 21.
    Lai JY, Lin PK, Hsiue GH, Cheng HY, Huang SJ, Li YT. Low bloom strength gelatin as a carrier for potential use in retinal sheet encapsulation and transplantation. Biomacromolecules. 2009;10(2):310–9. doi: 10.1021/bm801039n.CrossRefGoogle Scholar
  22. 22.
    Petrenko YA, Ivanov RV, Petrenko AY, Lozinsky VI. Coupling of gelatin to inner surfaces of pore walls in spongy alginate-based scaffolds facilitates the adhesion, growth and differentiation of human bone marrow mesenchymal stromal cells. J Mater Sci Mater Med. 2011;22(6):1529–40. doi: 10.1007/s10856-011-4323-6.CrossRefGoogle Scholar
  23. 23.
    Davis JM, ed. Basic cell culture. 2nd ed. Oxford University Press: Oxford, UK. 2002.Google Scholar
  24. 24.
    Yudin VE, et al. Wet spinning of fibers made of chitosan and chitin nanofibrils. Carbohydr Polym. 2014;108:176–82. doi: 10.1016/j.carbpol.2014.02.090.CrossRefGoogle Scholar
  25. 25.
    Kulichikhin VG, Semakov AV, Karbushev VV, Platé NA, Picken SJ. The chaos-to-order transition in critical modes of shearing for polymer and nanocomposite melts. Poly Sci Series A. 2009;51(11):1303–12. doi: 10.1134/S0965545X09110169.CrossRefGoogle Scholar
  26. 26.
    Yin H, Mo D, Chen D. Orientation behavior of attapulgite nanoparticles in poly(acrylonitrile)/attapulgite solutions by rheological analysis. J Poly Sci Part B: Polym Phys. 2009;47(10):945–54. doi: 10.1002/polb.21701.CrossRefGoogle Scholar
  27. 27.
    Balaban NQ, et al. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat Cell Biol. 2001;3(5):466–72. doi: 10.1038/35074532.CrossRefGoogle Scholar
  28. 28.
    Okuyama K, Noguchi K, Hanafusa Y, Osawa K, Ogawa K. Structural study of anhydrous tendon chitosan obtained via chitosan/acetic acid complex. Int J Biol Macromol. 1999;26(4):285–93.CrossRefGoogle Scholar
  29. 29.
    Okuyama K, Noguchi K, Kanenari M, Egawa T, Osawa K, Ogawa K. Structural diversity of chitosan and its complexes. Carbohydr Poly. 2000;41(3):237–47. doi: 10.1016/S0144-8617(99)00142-3.CrossRefGoogle Scholar
  30. 30.
    Zhang Y, Xue C, Xue Y, Gao R, Zhang X. Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction. Carbohydr Res. 2005;340(11):1914–7. doi: 10.1016/j.carres.2005.05.005.CrossRefGoogle Scholar
  31. 31.
    Rinaudo M. Chitin and chitosan: properties and applications. Prog Polym Sci. 2006;31(7):603–32. doi: 10.1016/j.progpolymsci.2006.06.001.CrossRefGoogle Scholar
  32. 32.
    Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials. 2003;24(24):4337–51.CrossRefGoogle Scholar
  33. 33.
    Zaborowska M, Bodin A, Backdahl H, Popp J, Goldstein A, Gatenholm P. Microporous bacterial cellulose as a potential scaffold for bone regeneration. Acta Biomater. 2010;6(7):2540–7. doi: 10.1016/j.actbio.2010.01.004.CrossRefGoogle Scholar
  34. 34.
    Hillberg AL, Holmes CA, Tabrizian M. Effect of genipin cross-linking on the cellular adhesion properties of layer-by-layer assembled polyelectrolyte films. Biomaterials. 2009;30(27):4463–70. doi: 10.1016/j.biomaterials.2009.05.026.CrossRefGoogle Scholar
  35. 35.
    Mendelsohn JD, Yang SY, Hiller J, Hochbaum AI, Rubner MF. Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films. Biomacromolecules. 2003;4(1):96–106. doi: 10.1021/bm0256101.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Victoria V. Kiroshka
    • 1
  • Valentina A. Petrova
    • 2
  • Daniil D. Chernyakov
    • 2
  • Yulia O. Bozhkova
    • 1
  • Katerina V. Kiroshka
    • 1
  • Yulia G. Baklagina
    • 2
  • Dmitry P. Romanov
    • 3
  • Roman V. Kremnev
    • 4
  • Yury A. Skorik
    • 2
  1. 1.Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of UkraineKharkovUkraine
  2. 2.Institute of Macromolecular Compounds of the Russian Academy of SciencesSt. PetersburgRussian Federation
  3. 3.Institute of Silicate Chemistry of the Russian Academy of SciencesSt. PetersburgRussian Federation
  4. 4.Institute of ChemistrySt. Petersburg State UniversitySt. PetersburgRussian Federation

Personalised recommendations