Hyaluronic acid stimulates the formation of calcium phosphate on CoCrMo alloy in simulated physiological solution

  • Ingrid Milošev
  • Julija Hmeljak
  • Andrej Cör


The behaviour of CoCrMo alloy has been studied in two simulated physiological solutions—NaCl and Hanks’ solutions—each containing the sodium salt of hyaluronic acid. Hyaluronic acid is a component of synovial joint fluid, so the behaviour of orthopaedic alloys in its presence needs to be assessed. Electrochemical methods, X-ray photoelectron spectroscopy and scanning electron microscopy have been used to analyse the composition, thickness and morphology of any layers formed on the alloy. The addition of hyaluronic acid shifts the corrosion potential and increases the value of polarization resistance. The presence of hyaluronic acid in simulated Hanks’ physiological solution stimulates the formation of a calcium phosphate layer, opening up the possibility for tailoring the surface properties of CoCrMo alloy. The viability of human osteoblast-like was determined using the Alamar® Blue Assay, while the osteogenic activity was evaluated by alkaline phosphatase activity. The presence of hyaluronic acid affects the alkaline phosphatase activity.


Hyaluronic Acid Calcium Phosphate Passive Film Immersion Time High Binding Energy 
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.



The authors thank Mojca Seručnik, BSc, and Gregor Žerjav, BSc, for valuable experimental work, Gregor Kapun, BSc, of the National Institute of Chemistry for expertise in the FE-SEM analysis, Dr. Janez Kovač for valuable comments on XPS measurements, and dr. Antonija Lesar for fruitful discussions. Human osteosarcoma (HOS), a human osteoblast-like cell line, was kindly donated by prof. Janja Marc, Faculty of Pharmacy, University of Ljubljana. Financial support by the Slovenian Research Agency is acknowledged (Grants No. J1-2243 and J1-4136).


  1. 1.
    Kasemo B. Biological surface science. Surface Sci. 2002;500:656–77.CrossRefGoogle Scholar
  2. 2.
    Puleo DA, Nanci A. Understanding and controlling the bone-implant interface. Biomaterials. 1999;20:2311–21.CrossRefGoogle Scholar
  3. 3.
    Xu T, Zhan N, Nichols HL, Shi D, Wen X. Modification of nanostructured materials for biomedical apllications. Mater Sci Eng C. 2007;27:579–94.CrossRefGoogle Scholar
  4. 4.
    Paital SR, Dahotre NB. Calcium phosphate coatings for bio-implant applications: materials, performance factors and methodologies. Mater Sci Eng R. 2009;66:1–70.CrossRefGoogle Scholar
  5. 5.
    Igual Muňoz A, Mischler S. Interactive effects of albumin and phosphate ions on the corrosion of CoCrMo implant alloy. J Electrochem Soc 2007;154: C562-70.Google Scholar
  6. 6.
    Hodgson AWE, Kurz S, Virtanen S, Fervel V, Olsson C-OA, Mischler S. Passive and transpassive behaviour of CoCrMo in simulated biological solutions. Electrochim Acta. 2004;49:2167–78.CrossRefGoogle Scholar
  7. 7.
    Li Y-S, Wang K, He P, Huang BX, Kovacs P. Surface-enhanced Raman spectroelectrochemical studies of corrosion films on implant Co–Cr–Mo alloy in biosimulating solutions. J Raman Spectrosc. 1999;30:97–103.CrossRefGoogle Scholar
  8. 8.
    Milošev I, Strehblow HH. The composition of the surface passive film formed on CoCrMo alloy in simulated physiological solution. Electrochim Acta. 2003;48:2767–74.CrossRefGoogle Scholar
  9. 9.
    Milošev I. Surface treatments for biomedical applications. In: Djokic Stojan S, editor. Modern aspects of electrochemistry, vol 55. Springer; 2012. p. 1–72.Google Scholar
  10. 10.
    Hanawa T, Hiromoto S, Asami K. Characterization of the surface oxide film of a Co-Cr-Mo alloy after being located in quasi-biological environments using XPS. Appl Surf Sci. 2001;183:68–75.CrossRefGoogle Scholar
  11. 11.
    Contu F, Elsener B, Böhni H. Characterization of implant materials in fetal bovine serum and sodium sulfate by electrochemical impedance spectroscopy. I Mechanically polished samples. J Biomed Mater Res. 2002;62:412–21.CrossRefGoogle Scholar
  12. 12.
    Hiromoto S, Noda K, Hanawa T. Development of electrolytic cell with cell-culture for metallic biomaterials. Corrosion Sci. 2002;44:955–65.CrossRefGoogle Scholar
  13. 13.
    Cheng X, et al. Corrosion behaviour of titanium in the presence of calcium phosphate and serum proteins. Biomaterials. 2005;26:7350–6.CrossRefGoogle Scholar
  14. 14.
    Milošev I. The effect of biomolecules on the behaviour of CoCrMo alloy in various simulated physiological solutions. Electrochim Acta. 2012;78:259–73.CrossRefGoogle Scholar
  15. 15.
    Yadav KL, Brown PW. Formation of hydroxyapatite in water, Hank’s solution, and serum at physiological temperature. J Biomed Mater Res A. 2003;65A:158–63.CrossRefGoogle Scholar
  16. 16.
    Day AJ, Sheenan JK. Hyaluronan: polysaccharide chaos to protein organisation. Curr Opin Chem Biol. 2001;11:617–22.Google Scholar
  17. 17.
    Barbucci R, Lamponi S, Borzacchiello A, Ambrosio L, Fini M, Torricelli P, Giardino R. Hyaluronic acid hydrogel in the treatment of osteoarthritis. Biomaterials. 2002;23:4503–13.CrossRefGoogle Scholar
  18. 18.
    Gribbon P, Heng BC, Hardingham TE. The analysis of intermolecular interactions in concnetrated hyaluronan solutions suggest no evidence for chain–chain association. Biochem J. 2000;350:329–35.CrossRefGoogle Scholar
  19. 19.
    Sheehan JK, Atkins EDT. X-ray fibre diffraction study of conformational changes in hyaluronate induced in the presence of sodium, potassium and calcium cations. Int J Biol Macromol. 1983;5:215–21.CrossRefGoogle Scholar
  20. 20.
    Collis JJ, Embery G. Adsorption of glycosaminoglycans to commercially pure titanium. Biomaterials. 1992;13:548–52.CrossRefGoogle Scholar
  21. 21.
    Ellingsen JE. A study on the mechanism of protein adsorption to TiO2. Biomaterials. 1991;12:593–6.CrossRefGoogle Scholar
  22. 22.
    Hanawa T, Ota M. Calcium phosphate naturally formed on titanium in electrolyte solution. Biomaterials 1991;12:767-14.Google Scholar
  23. 23.
    Briggs D, Rivière JC. Spectral interpretation. In: Briggs D, Seah MP, editors. Practical surface analysis by XPS and AES. New York: Chichester; 1990.Google Scholar
  24. 24.
    Seah MP. Summary of ISO/TC 201 standard: iSO 14701:–surface chemical analysis–X-ray photoelectron spectroscopy–measurement of silicon oxide thickness. Surf Interface Anal. 2012;44:876–8.CrossRefGoogle Scholar
  25. 25.
    Pan J, Thierry D, Leygraf C. Electrochemical impedance spectroscopy study of the passive film on titanium for implant applications. Electrochim Acta. 1996;41:1143–53.CrossRefGoogle Scholar
  26. 26.
    Aziz-Kerrzo, Conroy KG, Fenelon AM, Farrell ST, Breslin CB. Electrochemical studies on the stability and corrosion resistance of titanium-based implant materials. Biomaterials 2001;21:1531-9.Google Scholar
  27. 27.
    Souto RA, Laz MM, Reis RL. Degradation characteristics of hydroxyapatite coatings on orthopedic TiAlV in simulated physiological media investigated by electrochemical impedance spectroscopy. Biomaterials. 2003;24:4213–21.CrossRefGoogle Scholar
  28. 28.
    Hodgson AWE, Mueller Y, Forster D, Virtanen S. Electrochemical characterisation of passive films on Ti alloys under simulated biological conditions. Electrochim Acta. 2002;47:1913–23.CrossRefGoogle Scholar
  29. 29.
    Tamilselvi S, Raman V, Rajendran N. Corrosion behaviour of Ti-6Al-7Nb and Ti-6Al-4 V ELI alloys in the simulated body fluid solution by electrochemical impedance spectroscopy. Electrochim Acta. 2006;52:839–46.CrossRefGoogle Scholar
  30. 30.
    Metikoš-Huković M, Pilić Z, Babić R, Omanović D. Influence of alloying elements on the corrosion stability of CoCrMo implant alloy in Hank’s solution. Acta Biomater. 2006;2:693–700.CrossRefGoogle Scholar
  31. 31.
    Milošev I, Blejan D, Varvara S, Muresan LM. Effect of anodic oxidation on the corrosion behaviour of Ti-based materials in simulated physiological solution. submitted.Google Scholar
  32. 32.
    Wagner CD, Naumkin AV, Kraut-Vass A, Allison JW, Powell CJ, Rumble JR Jr. NIST X-ray photoelectron spectroscopy database, NIST standard reference database 20, Version 3.5, Data compiled and evaluated.
  33. 33.
    Tsutsumi Y, Nishimura D, Doi H, Nomura H, Hanawa T. Difference in surface reactions between titanium and zirconium in Hank’s solution to elucidate mechanism of calcium phosphate formation on titanium using XPS and cathodic polarization. Mat Sci Eng C 2009; 29:1702-8.Google Scholar
  34. 34.
    Frauchiger L, Taborelli M, Aronsson B-O, Descouts P. Ion adsorption on titanium surfaces exposed to a physiological solution. Appl Surf Sci. 1999;143:67–77.CrossRefGoogle Scholar
  35. 35.
    Milošev I, Jovanović Ž, Bajat JB, Jančić-Heinemann J, Mišković-Stanković VB. Surface analysis and electrochemical behaviour of aluminium pretreated by vinyltriethoxysilane films in mild NaCl solution. J Electrochem Soc. 2012;159:C303–11.Google Scholar
  36. 36.
    Feng B, Chen J, Zhang X. Interaction of calcium and phosphate in apatite coating on titanium with serum albumin. Biomaterials. 2002;23:2499–507.CrossRefGoogle Scholar
  37. 37.
    Hughes Wassell DT, Graham E. Adsorption of bovine serum albumin on to titanium powder. Biomaterials 1196;17:859-64.Google Scholar
  38. 38.
    Steinberg D, Klinger A, Kohavi D, Sela MN. Adsorption of human salivary proteins to tittanium powder I: adsortpion of human salivary albumin. Biomaterials. 1995;16:1339–43.CrossRefGoogle Scholar
  39. 39.
    Hay DI, Moreno EC. Differential adsorption and chemical affinities of protein for apatitic surfaces. J Dent Res. 1979;58:930–40.CrossRefGoogle Scholar
  40. 40.
    Sheenan JK, Brass A, Almond A. The conformations of hyaluronan in aqueous solution: comparison of theory and experiment. Biochem Soc Trans. 1999;27:121–4.Google Scholar
  41. 41.
    Almond A, Brass A, Sheenan JK. Deducing polymeric structure from aqueous molecular dynamics simulations of oligosaccharides: predictions from simulations of hyaluronan tetrasacccharides compared with hydrodynamic and X-ray fibre diffraction data. J Mol Biol. 1998;284:1425–37.CrossRefGoogle Scholar
  42. 42.
    Gu Z, Cai Q, He Y, Fu T, Li F. Degradation of hyaluronan by an electrochemical process. Carbohydr Polym. 2010;82:521–3.CrossRefGoogle Scholar
  43. 43.
    Taylor KR, Trowbridge JM, Rudisill JA, Termeer CC, Simon JC, Galo RL. Hyaluronan fragments stimulate endothelial recognition of injury through TLR4. J Biol Chem. 2004;279:17079–84.CrossRefGoogle Scholar
  44. 44.
    Scheibner S, Lutz MA, Boodoo S, Fenton MJ, Powell JD, Horton MR. Hyaluronan fragments act as an endogenous danger signal by engaging TLR2. J Immun. 2006;177:1272–81.Google Scholar
  45. 45.
    West DC, Hampson IN, Arnold F, Kumar S. Angiogenesis induced by degradation productts of hyaluronic acid. Science. 1985;228:1324–6.CrossRefGoogle Scholar
  46. 46.
    Bhakta G, Rai B, Lim ZXH, Hui JH, Stein GS, van Wijnen AJ, Nurcombe V, Prestwich GD, Cool SM. Hyluronic acid-based hydrogels functionalized with heparin that support controlled release of bioactive BMP-2. Biomaterials. 2012;33:6113–22.CrossRefGoogle Scholar
  47. 47.
    Huang Y, Luo Q, Li X, Zhang F, Zhao S. Fabrication and in vitro evaluation of the collagen/hyaluronic acid PEM coating crosslinked with functionalized RGD peptide on titanium. Acta Biomateriala. 2012;8:866–77.CrossRefGoogle Scholar
  48. 48.
    Chua P-H, Neoh K-G, Kang E-T, Wang W. Surface functionalization of titanium with hyaluronic acid/chitosan polelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion. Biomaterials. 2008;29:1412–21.CrossRefGoogle Scholar
  49. 49.
    Park JK, Shim JH, Kang KS, Yeom J, Jung HS, Kim JY, Lee KH, Kim TH, Kim SY, Cho DW, Hahn SK. Solid free-form fabrication of tissue engineering scaffolds with a poly(lactic-co-glycolic) grafted hyaluronic acid conjugate encapsulating an intact bone morphogenetic protein-2/poly(ethylene glycol) complex. Adv Funct Mater. 2011;21:2906–12.CrossRefGoogle Scholar
  50. 50.
    Jiao Y, Liu Z, Shao X, Zhou C. Protein adsorption and cytocompatibility of poly(l-lactic acid) surfaces modified with biomacromolecules. J Appl Polymer Sci. 2012;125:E501–10.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Ingrid Milošev
    • 1
    • 2
  • Julija Hmeljak
    • 3
  • Andrej Cör
    • 2
    • 3
  1. 1.Department of Physical and Organic ChemistryJožef Stefan InstituteLjubljanaSlovenia
  2. 2.Valdoltra Orthopaedic HospitalAnkaranSlovenia
  3. 3.Faculty of Health SciencesUniversity of PrimorskaIzolaSlovenia

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