Skip to main content

Collagen-Chondroitin Sulphate-Hydroxyapatite Porous Composites: A Histochemical and Electron Microscopy Approach

  • Otilia Zarnescu (a1), Oana Craciunescu (a2) and Lucia Moldovan (a2)

In this study the structure of collagen-chondroitin sulphate-hydroxyapatite porous composites is investigated by histochemical (Von Kossa staining), immunohistochemical, and transmission electron microscopy. Examination of composites on picrosirius red stained sections showed that polarization colors of collagen were generally in the range of orange-red. Immunofluorescence data indicate that chondroitin sulphate was either chemically incorporated into the bulk structure of collagen scaffolds or attached on surfaces of collagen bundles. Depending on the ratio between collagen:chondroitin sulphate:hydroxyapatite, von Kossa histochemical staining showed a progressive loading of collagen-chondroitin sulphate bundles with hydroxyapatite. Transmission electron microscopy studies have shown that composites contain mostly collagen fibrils aggregated with random orientation with very few collagen fibers showing the 67-nm banding pattern. Hydroxyapatite deposits of various sizes occurred among the collagen fibrils.

Corresponding author
Corresponding author. E-mail:
Hide All
Burger, M., Sherman, B.S. & Sobel, A.E. (1962). Observations of the influence of chondroitin sulphate on the rate of bone repair. J Bone Joint Surg 44B, 675687.
Dayan, D., Hiss, Y., Hirshberg, A., Bubis, J.J. & Wolman, M. (1989). Are the polarization colors of picrosirius red-stained collagen determined only by the diameter of the fibers? Histochemistry 93, 2729.
Declercq, H., Van Der Vreken, N., De Maeyer, E., Verbeeck, R., Schacht, E., De Ridder, L. & Cornelissen, M. (2004). Isolation, proliferation and differentiation of osteoblastic cells to study cell/biomaterial interactions: Comparison of different isolation techniques and source. Biomaterials 25, 757768.
Glimcher, M.J. (1992). The nature of the mineral component of bone and the mechanism of calcification. In Metabolic Bone Disease and Clinically Related Disorders, Coe, F.L. & Favus, M.J. (Eds.), pp. 265286. New York: Raven Press Ltd.
Holbrook, K.A. & Smith, L.T. (1993). Morphology of connective tissue: Structure of the skin and tendon. In Connective Tissue and Its Heritable Disorders—Molecular, Genetic and Medical Aspects, Royce, P.M. & Steinmann, B. (Eds.), pp. 5171. New York: Wiley-Liss.
Junqueira, L.C., Cossermelli, W. & Brentani, R. (1978). Differential staining of collagens type I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn 41, 267274.
Kamakura, S., Sasaki, K., Honda, Y., Anada, T., Matsui, K., Echigo, S. & Suzuki, O. (2007). Dehydrothermal treatment of collagen influences on bone regeneration by octacalcium phosphate (OCP) collagen composites. J Tissue Eng Regen Med 1, 450456.
Kikuchi, M., Ikoma, T., Itoh, S., Matsumoto, H.N., Koyama, Y., Takakuda, K., Shinomiya, K. & Tanaka, J. (2004). Biomimetic synthesis of bone-like nanocomposites using the self organization mechanism of hydroxyapatite and collagen. Compos Sci Technol 64, 819825.
Liu, C., Han, Z. & Czernuszka, J.T. (2009). Gradient collagen/nanohydroxyapatite composite scaffold: Development and characterization. Acta Biomater 5, 661669.
Moldovan, L., Craciunescu, O., Oprita, E.I., Balan, M. & Zarnescu, O. (2009). Collagen-chondroitin sulfate-hydroxyapatite porous composites: Preparation, characterization and in vitro biocompatibility testing. Roum Biotechnol Lett 14, 44624469.
Moldovan, L., Oancea, O., Zarnescu, O. & Barboi, G. (1996). In vitro biocompatibility evaluation of collagen-based sponges. Rev Roum Biol Biol Anim 4, 5964.
Murugan, R. & Ramakrishna, S. (2005). Development of nanocomposites for bone grafting. Comp Sci & Tech 65, 23852406.
Rhee, S.H. & Tanaka, J. (2002). Self-assembly phenomenon of hydroxyapatite nanocrystals on chondroitin sulfate. J Mater Sci Mater Med 13, 597600.
Supová, M. (2009). Problem of hydroxyapatite dispersion in polymer matrices: A review. J Mater Sci Mater Med 20, 1201–213.
Suvorova, E.I., Klechkovskaya, V.V., Komarov, V.F., Severin, A.V., Melikhov, I.V. & Buffat, P.A. (2006). Electron microscopy of biomaterials based on hydroxyapatite. Crystallogr Rep 51, 881887.
Weiner, S. & Traub, W. (1986). Organization of hydroxyapatite crystals within collagen fibrils. FEBS Lett 206, 262266.
Xie, J., Baumann, M.J. & McCabe, L.R. (2004). Osteoblasts respond to hydroxyapatite surfaces with immediate changes in gene expression. J Biomed Mater Res A 71, 108117.
Yunoki, S., Marukawa, E., Ikoma, T., Sotome, S., Fan, H., Zhang, X., Shinomiya, K. & Tanaka, J. (2007). Effect of collagen fibril formation on bioresorbability of hydroxyapatite/collagen composites. J Mater Sci Mater Med 18, 21792183.
Zhang, S.M., Cui, F.Z., Liao, S.S., Zhu, Y. & Han, L. (2003). Synthesis and biocompatibility of porous nano-hydroxyapatite/collagen/alginate composite. J Mater Sci Mater Med 14, 641645.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Full text views

Total number of HTML views: 8
Total number of PDF views: 29 *
Loading metrics...

Abstract views

Total abstract views: 194 *
Loading metrics...

* Views captured on Cambridge Core between September 2016 - 19th July 2018. This data will be updated every 24 hours.