Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS

  • A. R. BoydEmail author
  • G. A. Burke
  • H. Duffy
  • M. Holmberg
  • C. O’ Kane
  • B. J. Meenan
  • P. Kingshott


Protein adsorption onto calcium phosphate (Ca–P) bioceramics utilised in hard tissue implant applications has been highlighted as one of the key events that influences the subsequent biological response, in vivo. This work reports on the use of surface-matrix assisted laser desorption ionisation mass spectrometry (Surface-MALDI-MS) as a technique for the direct detection of foetal bovine serum (FBS) proteins adsorbed to hybrid calcium phosphate/titanium dioxide surfaces produced by a novel radio frequency (RF) magnetron sputtering method incorporating in situ annealing between 500°C and 700°C during deposition. XRD and XPS analysis indicated that the coatings produced at 700°C were hybrid in nature, with the presence of Ca–P and titanium dioxide clearly observed in the outer surface layer. In addition to this, the Ca/P ratio was seen to increase with increasing annealing temperature, with values of between 2.0 and 2.26 obtained for the 700°C samples. After exposure to FBS solution, surface-MALDI-MS indicated that there were significant differences in the protein patterns as shown by unique peaks detected at masses below 23.1 kDa for the different surfaces. These adsorbates were assigned to a combination of growth factors and lipoproteins present in serum. From the data obtained here it is evident that surface-MALDI-MS has significant utility as a tool for studying the dynamic nature of protein adsorption onto the surfaces of bioceramic coatings, which most likely plays a significant role in subsequent bioactivity of the materials.


Protein Adsorption Hybrid Coating Titanium Layer Post Deposition Annealing ICDD File 
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.



This work was kindly supported by a RTD Networking grant from Invest Northern Ireland (RTD 375). This work was part of the Centre for Nanostructured Polymer Surfaces for Medical Applications, funded by the Danish Ministry for Science, Technology and Innovation (2002-603-4001-87). The authors would also like to thank Kratos Analytical (UK) for their assistance with the XPS analysis.


  1. 1.
    Wolke JGC, van Dijk K, Schaeken HG, de Groot K, Jansen JA. Evaluation of plasma-spray and magnetron-sputter Ca–P-coated implants: an in vivo experiment using rabbits. J Biomed Mater Res. 1994;28:1477–84.CrossRefGoogle Scholar
  2. 2.
    van Dijk K, Schaeken HG, Wolke JGC, Maree CHM, Habraken FHPM, Verhoeven J, Jansen JA. Enhancing osseointegration using surface-modified titanium implants. J Biomed Mater Res. 1995;29:269–76.CrossRefGoogle Scholar
  3. 3.
    van Dijk K, Schaeken HG, Maree CHM, Verhoeven J, Wolke JGC, Habraken FHPM, Jansen JA. Influence of Ar pressure on r.f. magnetron-sputtered Ca5(PO4)3OH layers. Surf Coat Technol. 1995;76–77:206–10.CrossRefGoogle Scholar
  4. 4.
    van Dijk K, Schaeken HG, Wolke JGC, Jansen JA. Influence of annealing temperature on RF magnetron sputtered calcium phosphate coatings. Biomaterials. 1996;17:405–10.CrossRefGoogle Scholar
  5. 5.
    van Dijk K, Verhoeven J, Maree CHM, Habraken FHPM, Jansen JA. Study of the influence of oxygen on the composition of thin films obtained by r.f. sputtering from a Ca5(PO4)3 OH target. Thin Solid Films. 1997;304:191–5.CrossRefGoogle Scholar
  6. 6.
    Wolke JGC, de Groot K, Jansen JA. Dissolution and adhesion behaviour of radio-frequency magnetron-sputtered Ca–P coatings. J Mater Sci. 1998;33:3371–6.CrossRefGoogle Scholar
  7. 7.
    Boyd AR, Akay M, Meenan BJ. Influence of target surface degradation on the properties of r.f. magnetron-sputtered calcium phosphate coatings. Surf Int Anal. 2003;35:188–98.CrossRefGoogle Scholar
  8. 8.
    Wolke JGC, van der Waerden JPCM, Schaeken HG, Jansen JA. In vivo dissolution behavior of various RF magnetron-sputtered Ca–P coatings on roughened titanium implants. Biomaterials. 2003;24:2623–9.CrossRefGoogle Scholar
  9. 9.
    Nelea V, Morosanu C, Iliescu M, Mihailescu IN. Microstructure and mechanical properties of hydroxyapatite thin films grown by RF magnetron sputtering. Surf Coat Technol. 2003;173:315–22.CrossRefGoogle Scholar
  10. 10.
    Boyd AR, Meenan BJ, Leyland NS. Surface characterisation of the evolving nature of radio frequency (RF) magnetron sputter deposited calcium phosphate thin films after exposure to physiological solution. Surf Coat Technol. 2006;200:6002–13.CrossRefGoogle Scholar
  11. 11.
    Meenan BJ, Boyd A, Leyland NS, Love E, Akay M. The influence of substrate morphology on the structure and composition of RF sputter deposited calcium phosphate thin films. Bioceramics. 1999;12:471–4.Google Scholar
  12. 12.
    Lo WJ, Grant DM, Ball MD, Welsh BS, Howdle SM, Antonov EN, Bagratashvili VN, Popov VK. Physical, chemical, and biological characterization of pulsed laser deposited and plasma sputtered hydroxyapatite thin films on titanium alloy. J Biomed Mater Res. 2000;50:536–45.CrossRefGoogle Scholar
  13. 13.
    Boyd AR, Duffy H, McCann R, Cairns ML, Meenan BJ. The Influence of argon gas pressure on co-sputtered calcium phosphate thin films. Nucl Instrum Methods Phys Res B. 2007;258:421–8.CrossRefGoogle Scholar
  14. 14.
    Cairns ML, Meenan BJ, Burke GA, Boyd AR. Effect of nanoscale topography on fibronectin adsorption to sputter deposited calcium phosphate thin films. Int J Nano Biomater. 2008;1:280–98.CrossRefGoogle Scholar
  15. 15.
    Lu Y, Li M, Li S, Wang Z, Zhu R. Plasma-sprayed hydroxyapatite + titania composite bond coat for hydroxyapatite coating on titanium substrate. Biomaterials. 2004;25:4393–403.CrossRefGoogle Scholar
  16. 16.
    Li H, Khor KA, Cheang P. Impact formation and microstructure characterization of thermal sprayed hydroxyapatite/titania composite coatings. Biomaterials. 2003;24:949–57.CrossRefGoogle Scholar
  17. 17.
    Lin C, Yen S. Characterization and bond strength of electrolytic HA/TiO2 double layers for orthopedic applications. J Mater Sci Mater Med. 2004;15:1237–46.CrossRefGoogle Scholar
  18. 18.
    Wang Y, Li Y, Yu H, Ding J, Tang X, Li J, Zhou Y. In situ fabrication of bioceramic composite coatings by laser cladding. Surf Coat Technol. 2005;200:2080–4.CrossRefGoogle Scholar
  19. 19.
    Manso M, Langlet M, Fernandez M, Vasquez L, Martinez-Duart JM. Surface and interface analysis of hydroxyapatite/TiO2 biocompatible structures. Mater Sci Eng C. 2003;23:451–4.CrossRefGoogle Scholar
  20. 20.
    Milella E, Conentino F, Licciulli A, Massaro C. Preparation and characterisation of titania/hydroxyapatite composite coatings obtained by sol–gel process. Biomaterials. 2001;22:1425–31.CrossRefGoogle Scholar
  21. 21.
    Kumar RR, Wang M. Functionally graded bioactive coatings of hydroxyapatite/titanium oxide composite system. Mater Lett. 2002;55:133–7.CrossRefGoogle Scholar
  22. 22.
    Boyd AR, Burke GA, Duffy H, Cairns ML, O’Hare P, Meenan BJ. Characterisation of calcium phosphate/titanium dioxide hybrid coatings. J Mater Sci Mater Med. 2008;19:485–98.CrossRefGoogle Scholar
  23. 23.
    Boyd AR, Duffy H, McCann R, Meenan BJ. Sputter deposition of calcium phosphate/titanium dioxide hybrid thin films. Mater Sci Eng C. 2008;28:228–36.CrossRefGoogle Scholar
  24. 24.
    Rosengren Å, Pavlovic E, Oscarsson S, Krajewski A, Ravaglioli A, Piancastelli A. Plasma protein adsorption pattern on characterized ceramic biomaterials. Biomaterials. 2002;23:1237–47.CrossRefGoogle Scholar
  25. 25.
    Rees SG, Hughes Wassell DT, Shellis RP, Embery G. Effect of serum albumin on glycosaminoglycan inhibition of hydroxyapatite formation. Biomaterials. 2004;25:971–7.CrossRefGoogle Scholar
  26. 26.
    Siebers MC, ter Brugge PJ, Walboomers XF, Jansen JA. Integrins as linker proteins between osteoblasts and bone replacing materials. A critical review. Biomaterials. 2005;26:137–46.CrossRefGoogle Scholar
  27. 27.
    Sawyer AA, Hennessy KM, Bellis SL. Regulation of mesenchymal stem cell attachment and spreading on hydroxyapatite by RGD peptides and adsorbed serum proteins. Biomaterials. 2005;26:1467–75.CrossRefGoogle Scholar
  28. 28.
    Sawyer AA, Hennessy KM, Bellis SL. The effect of adsorbed serum proteins, RGD and proteoglycan-binding peptides on the adhesion of mesenchymal stem cells to hydroxyapatite. Biomaterials. 2007;28:383–92.CrossRefGoogle Scholar
  29. 29.
    Kilpadi KL, Chang P-L, Bellis SL. Hydroxylapatite binds more serum proteins, purified integrins, and osteoblast precursor cells than titanium or steel. J Biomed Mater Res A. 2001;57:258–67.CrossRefGoogle Scholar
  30. 30.
    Serro AP, Bastos M, Costa Pessoa J, Saramago B. Bovine serum albumin conformational changes upon adsorption on titania and on hydroxyapatite and their relation with biomineralization. J Biomed Mater Res A. 2004;70A:420–7.CrossRefGoogle Scholar
  31. 31.
    Ellingsen JE. A study on the mechanism of protein adsorption to TiO2. Biomaterials. 1991;12:593–6.CrossRefGoogle Scholar
  32. 32.
    Yang Y, Glover R, Ong JL. Fibronectin adsorption on titanium surfaces and its effect on osteoblast precursor cell attachment. Colloid Surf B Biointerfaces. 2003;30:291–7.CrossRefGoogle Scholar
  33. 33.
    Klinger A, Steinberg D, Kohavi D, Sela MN. Mechanism of adsorption of human albumin to titanium in vitro. J Biomed Mater Res A. 1997;36:387–92.CrossRefGoogle Scholar
  34. 34.
    Yang Y, Cavin R, Ong JL. Protein adsorption on titanium surfaces and their effect on osteoblast attachment. J Biomed Mater Res A. 2003;67A:344–9.CrossRefGoogle Scholar
  35. 35.
    Wilson CJ, Clegg RE, Leavesley DI, Pearcy MJ. Mediation of biomaterial–cell interactions by adsorbed proteins: a review. Tissue Eng. 2005;11(1–2):1–18. (and the cited references).CrossRefGoogle Scholar
  36. 36.
    Omenn GS. Exploring the human plasma proteome. Proteomics. 2005;5:3223–5.CrossRefGoogle Scholar
  37. 37.
    Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics. 2002;1:845–67.CrossRefGoogle Scholar
  38. 38.
    Zheng X, Baker H, Hancock WS, Fawaz F, McCaman M, Pungor E Jr. Proteomic analysis for the assessment of different lots of fetal bovine serum as a raw material for cell culture. Part IV. Application of proteomics to the manufacture of biological drugs. Biotechnol Prog. 2006;22(5):1294–300.CrossRefGoogle Scholar
  39. 39.
    Kingshott P, Hoecker H. Adsorption of proteins: assessment methods. In: Somasundaran P, editor. Encyclopedia of surface and colloid science, vol 5. 2st ed. New York: Taylor and Francis; 2006. p. 669–94.Google Scholar
  40. 40.
    Villar-Garea A, Griese M, Imhof A. Biomarker discovery from body fluids using mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci. 2007;849(1–2):105–14.CrossRefGoogle Scholar
  41. 41.
    Zenobi R, Knochenmuss R. Ion formation in MALDI mass spectrometry. Mass Spectrom Rev. 1998;17:337–66.CrossRefGoogle Scholar
  42. 42.
    Kingshott P, St John HAW, Chatelier RC, Griesser HJ. Surface-MALDI mass spectrometry detection of proteins adsorbed in vivo onto contact lenses. J Biomed Mater Res. 2000;49:36–42.CrossRefGoogle Scholar
  43. 43.
    Griesser HJ, Kingshott P, McArthur SL, McLean KM, Kinsel GR, Timmons RB. Surface-MALDI mass spectrometry in biomaterials research. Biomaterials. 2004;25:4861–75.CrossRefGoogle Scholar
  44. 44.
    Konashi K, Kambara M, Noshi H, Uemura MJ. X-ray photoelectron spectroscopic (ESCA) study on the surface of hydroxyapatite. J Osaka Dent Univ. 1987;21:1–8.Google Scholar
  45. 45.
    Roome CM, Adam CD. Crystallite orientation, anisotropic strains in thermally sprayed hydroxyapatite coatings. Biomaterials. 1995;16:691–6.CrossRefGoogle Scholar
  46. 46.
    Tong W, Chen J, Li X, Feng J, Cao Y, Yang Z, Zhang X. Preferred orientation of plasma sprayed hydroxyapatite coatings. J Mater Sci. 1996;31:3739–42.CrossRefGoogle Scholar
  47. 47.
    Jha LJ, Santos JD, Knowles JC. Characterization of apatite layer formation on P2O5–CaO, P2O5–CaO–Na2O, and P2O5–CaO–Na2O–Al2O3 glass hydroxyapatite composites. J Biomed Mater Res. 1996;31:481–6.CrossRefGoogle Scholar
  48. 48.
    Ong JL, Lucas LC, Raikar GN, Weimer JJ, Gregory JC. Surface characterization of ion-beam sputter-deposited Ca–P coatings after in vitro immersion. Colloid Surf A Physiochem Eng Asp. 1994;87:151–62.CrossRefGoogle Scholar
  49. 49.
    Long JD, Xu S, Foo HY, Diong CH. Syntheses and properties of bioactive Ca–P–Ti thin films synthesized by reactive plasma co-sputtering deposition. Key Eng Mater. 2003;240–242:303–6.CrossRefGoogle Scholar
  50. 50.
    Long J, Xu S, Cai JW, Jiang N, Lu JH, Ostrikov KN, Diong CH. Structure, bonding state and in vitro study of Ca–P–Ti film deposited on Ti6Al4V by RF magnetron sputtering. Mater Sci Eng C Biomim Mater Sens Syst. 2002;20:175–80.CrossRefGoogle Scholar
  51. 51.
    Lee TM, Chang E, Yang CY. Surface characteristics of Ti6Al4V alloy: effect of materials, passivation and autoclaving. J Mater Sci Mater Med. 1998;9:439–48.CrossRefGoogle Scholar
  52. 52.
    Kumar PM, Badrinarayanan S, Sastry M. Nanocrystalline TiO2 studied by optical, FTIR and X-ray photoelectron spectroscopy: correlation to presence of surface states. Thin Solid Films. 2000;358:122–30.CrossRefGoogle Scholar
  53. 53.
    Xu S, Long J, Sim L, Diong CH, Ostrikov K (Ken). RF plasma sputtering deposition of hydroxyapatite bioceramics: synthesis, performance, and biocompatibility. Plasma process. Polymer. 2005;2:373–90.Google Scholar
  54. 54.
    Qian WJ, Jacobs JM, Camp DG II, Monroe ME, Moore RJ, Gritsenko MA, Calvano SE, Lowry SF, Xiao W, Moldawer LL, Davis RW, Tompkins RG, Smith RD. Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry. Proteomics. 2005;5:572–84.CrossRefGoogle Scholar
  55. 55.
    iProClass database.
  56. 56.
    Fujita M, Ishihara M, Ono K, Hattori H, Kurita A, Shimizu M, Mitsumaru A, Segawa D, Hinokiyama K, Kusama Y, Kikuchi M, Maehara T. Adsorption of inflammatory cytokines using a heparin-coated extracorporeal circuit. Artif Organs. 2000;26(12):1020–5.CrossRefGoogle Scholar
  57. 57.
    Yan X, Scherphof GL, Kamps JAAM. Liposome opsonization. J Lipsome Res. 2005;15:109–39.Google Scholar
  58. 58.
    Sun D-H, Trindade CD, Nakashima Y, Maloney WJ, Goodman SB, Schurman DJ, Smith RL. Human serum opsonization of orthopedic biomaterial particles: protein-binding and monocyte/macrophage. J Biomed Mater Res. 2003;65A:290–8.CrossRefGoogle Scholar
  59. 59.
    Cedervall T, Lynch I, Foy M, Berggærd T, Donnelly SC, Cagney G, Linse S, Dawson KA. Detailed identification of plasma proteins adsorbed on copolymer nanoparticles. Angew Chem Int Ed. 2007;46:5754–6.CrossRefGoogle Scholar
  60. 60.
    Banks RE, Stanley AJ, Cairns DA, Barrett JH, Clarke P, Thompson D, Selby PJ. Influences of blood sample processing on low-molecular-weight proteome identified by surface-enhanced laser desorption/ionization mass spectrometry. Clin Chem. 2005;51(9):1637–49.CrossRefGoogle Scholar
  61. 61.
    Zimmerman LJ, Wernke GR, Caprioli RM, Liebler DC. Identification of protein fragments as pattern features in MALDI-MS analysis of serum. J Proteome Res. 2005;4:1672–80.CrossRefGoogle Scholar
  62. 62.
    Kandori K, Tsuyama S, Tanaka H, Ishikawa T. Protein adsorption characteristics of calcium hydroxyapatites modified with pyrophosphoric acids. Colloids Surf B Biointerfaces. 2007;58:98–104.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • A. R. Boyd
    • 1
    Email author
  • G. A. Burke
    • 1
  • H. Duffy
    • 1
  • M. Holmberg
    • 2
  • C. O’ Kane
    • 1
  • B. J. Meenan
    • 1
  • P. Kingshott
    • 3
  1. 1.Nanotechnology and Integrated Bioengineering Centre (NIBEC), University of Ulster at JordanstownAntrimNorthern Ireland (UK)
  2. 2.Department of Micro- and NanotechnologyTechnical University of DenmarkRoskildeDenmark
  3. 3.Faculty of Science, The Interdisciplinary Nanoscience Centre (iNANO)University of AarhusAarhus CDenmark

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