The Study of Type I Collagen by Immunoblotting in Samples of Bone-Plastic Biomaterials

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T.N. Medvedeva
L.T. Volova
L.N. Kulagina


The type I collagen was studied in samples of two types of osteoplastic materials produced in the Biotech Research Institute of the Samara State Medical University using immunoblotting. The demineralized samples used in the work were compact bone powder and crushed material of human cancellous bone tissue. Collagen and its polypeptides were separated in a 5% polyacrylamide gel with 3.6 M urea according to the method of Hayashi and Nagai (1979). The advantage of the method is the separation under these conditions of type I and III collagen, as well as the α1(I) and α2(I) chains of type I collagen. Immunoblotting was carried out by diffusion method according to the method of Towbin et al. (1979) using nitrocellulose membranes (Santa Cruz, USA). Primary goat polyclonal antibodies to denatured collagen, 1:500 dilution (Millipore) were used. Peroxidase-conjugated secondary antibodies (mouse vs. goat), 1:80000 dilution (Sigma) were used also. It has been established that the bulk of the compact bone protein is localized between the α1- and α2-fractions of collagen. In samples of cancellous bone tissue, a molecular reduction of the protein is noted. Protein macromolecules with a gradually decreasing molecular weight and low molecular weight polypeptides migrating in the gel with a wide front up to the indicator line are detected. Due to the low specificity of osteoblast integrins in regenerating bone tissue, collagen polypeptides, as well as protein molecules retained in implants, can act as inducers of synthetic processes occurring in osteoblast nuclei. Protein fragmentation products in the implant can act as signaling molecules that trigger cascades of enzymatic reactions and intracellular signaling pathways.

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Medvedeva, T., Volova, L., & Kulagina, L. (2023). The Study of Type I Collagen by Immunoblotting in Samples of Bone-Plastic Biomaterials. Biomedical Chemistry: Research and Methods, 6(2), e00189.


  1. Glowacki, J., Mulliken, J.B. (1985) Demineralized bone implants. Clin. Plast. Surg., 12(2), 233-241.
  2. Goldberg, V.M., Stevenson, S. (1993) The biology of bone grafts. Semin. Arthroplasty, 4(2), 58-63.
  3. Karimbux, N.Y., Shirakian, A., Weber, H.P., Nishimura, I. (1995) A new animal model for molecular biological analysis of the implant-tissue interface: spatial expression of type XII collagen mRNA around a titanium oral implant. J. Oral Implantol., 21(2), 107-113.
  4. Forlino, A., Marini, J.C. (2016) Osteogenesis imperfecta. Lancet, 387(10028), 1657-1671. DOI
  5. Suzawa, M., Tamura, Y., Fukumoto, S., Miyazono, K., Fujita, T., Kato, S., Takeuchi, Y. (2002) Stimulation of Smad1 transcriptional activity by Ras-extracellular signal-regulated kinase pathway: a possible mechanism for collagen-dependent osteoblastic differentiation. J. Bone Miner. Res., 17(2), 240-248. DOI
  6. Volova, L.T., Krivoshchekov, E.P., Grigoriev, S.G., Krupyshev, I.A., Trunin, D.A. (1987) Procurement and preservation of biological tissues and their use in practical public health. Guidelines, KPO "ZIM", Kuibyshev ,19 p.
  7. Hayashi, T., Nagai, Y. (1979) Separation of the α chains of Type I and III collagens by SDS-polyacrylamide gel electrophoresis. J. Biochem., 86(2), 453-459.
  8. Towbin, H., Staehelin, T., Gordon, J. (1979) Electroforetic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA. Biochemistry, 76(9), 4350-4354.
  9. Dodge, G.R., Poole, A.R. (1989) Immunohistochemical detection and immunochemical analysis of type II collagen degradation in human normal, rheumatoid and osteoarthritic articular cartilages and in explants of bovine articular cartilage cultured with interleukin 1. J. Clin. Invest., 83, 647-661.
  10. Jansen, I.D.C., Papapoulos, S.E., Bravenboer, N., de Vries, T.J., Appelman-Dijkstra, N.M. (2021) Increased bone resorption during lactation in pycnodysostosis. Int. J. Mol. Sci., 22(4), 18. DOI
  11. Timchenko, E.V., Timchenko, P.E., Volova, L.T., Ponomareva, Yu.V., Taskina, L.A. (2014) Raman scattering study of the organomineral structure of bone implants. Quantum Electronics, 44(7), 696-699.
  12. Timchenko, E.V., Timchenko, P.E., Volova, L.T., Milyakova, M.N., Maksimenko, N.A., Taskina, L.A. (2015) Application of the Raman spectroscopy method to assess the demineralization of bone grafts during their preparation. Optical Journal, 82(3), 30-36.
  13. Kolsanov, A.V., Volova, L.T., Trunin, D.A., Popov, N.V., Ponomareva, Yu.V. (2017) Conducting preclinical trials of personalized bone implants using human cell cultures. Postgraduate Physician, 85(6.3), 367-377.
  14. Timchenko, P.E., Zakharov, V.P., Volova, L.T., Boltovskaya, V.V., Timchenko, E.V. (2011) Microscopic control of the implant osseointegration process. Computer Optics, 35(2), 183-188.
  15. Tsiklin, I.L., Pugachev, E.I., Kolsanov, A.V., Timchenko, E.V., Boltovskaya, V.V., Timchenko, P.E., Volova, L.T. (2022) Biopolymer material from human spongiose for regenerative medicine application. Polymers, 14(5), 941. DOI
  16. Fuller, G.M., Shields, D. (1998) Molecular Basis of Medical Cell Biology. First Edition. Appleton and Lange. Stamford, Connecticut.