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Calcified Tissue International

, Volume 103, Issue 5, pp 554–566 | Cite as

Deproteinization of Cortical Bone: Effects of Different Treatments

  • Frances Y. Su
  • Siyuan Pang
  • Yik Tung Tracy Ling
  • Peter Shyu
  • Ekaterina Novitskaya
  • Kyungah Seo
  • Sofia Lambert
  • Kimberlin Zarate
  • Olivia A. Graeve
  • Iwona Jasiuk
  • Joanna McKittrick
Original Research
  • 145 Downloads

Abstract

Bone is a biological composite material having collagen and mineral as its main constituents. In order to better understand the arrangement of the mineral phase in bone, porcine cortical bone was deproteinized using different chemical treatments. This study aims to determine the best method to remove the protein constituent while preserving the mineral component. Chemicals used were H2O2, NaOCl, NaOH, and KOH, and the efficacy of deproteinization treatments was determined by thermogravimetric analysis and Raman spectroscopy. The structure of the residual mineral parts was examined using scanning electron microscopy. X-ray diffraction was used to confirm that the mineral component was not altered by the chemical treatments. NaOCl was found to be the most effective method for deproteinization and the mineral phase was self-standing, supporting the hypothesis that bone is an interpenetrating composite. Thermogravimetric analyses and Raman spectroscopy results showed the preservation of mineral crystallinity and presence of residual organic material after all chemical treatments. A defatting step, which has not previously been used in conjunction with deproteinization to isolate the mineral phase, was also used. Finally, Raman spectroscopy demonstrated that the inclusion of a defatting procedure resulted in the removal of some but not all residual protein in the bone.

Keywords

Deproteinization Thermogravimetric analysis Cortical bone Raman spectroscopy Scanning electron microscopy 

Notes

Acknowledgements

This work was supported by a National Science Foundation Biomaterials Grants (1507978 and 1507169) and a Multi-University Research Initiative grant through the Air Force Office of Scientific Research (AFOSR-FA9550-15-1-0009). This work was performed in part at the San Diego Nanotechnology Infrastructure (SDNI) of UCSD, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (Grant ECCS-1542148). This study was partially funded by NSF REU Discoveries in Bioimaging (EEC 14-61038). We thank Ryan Anderson of the Nano3 Laboratory of CalIt2 for helping with the SEM and optical microscopy and Prof. Marc A. Meyers from UC San Diego for his kind and enthusiastic support of this project. We thank Prof. Henry Schwarcz from McMaster University for helpful comments about this manuscript. We also thank Dr. Julio Soares at Materials Research Lab for assistance in the Raman spectroscopy at the University of Illinois, and Tianqi Ren for help with X-ray diffraction at UC San Diego. We thank Joyce Mok for preliminary defatting experiments and Dr. Michael Frank and Jungmin Ha for their advice on phosphate indicators. Sofia Lambert and Kimberlin Zarate are thankful to the UC San Diego ENLACE program, which allowed them to participate in this research project. Yik Tung Tracy Ling would like to thank Dr. Marina Marjanovic and Joanne Li for running Discoveries in Bioimaging REU program in conjunction with SROP.

Compliance with Ethical Standards

Conflict of interest

Frances Y. Su, Siyuan Pang, Yik Tung Tracy Ling, Peter Shyu, Ekaterina Novitskaya, Kyungah Seo, Sofia Lambert, Kimberlin Zarate, Olivia A. Graeve, Iwona Jasiuk, and Joanna McKittrick have no conflicts of interest to declare.

Human and Animal Rights and Informed Consent

All applicable international, national, and institutional guidelines on animal care and use were followed.

Supplementary material

223_2018_453_MOESM1_ESM.docx (2 mb)
Supplementary material 1 (DOCX 2064 KB)

References

  1. 1.
    Weiner S, Wagner HD (1998) The material bone: Structure mechanical function relations. Annu Rev Mater Sci 28:271–298CrossRefGoogle Scholar
  2. 2.
    Ritchie RO (2011) The conflicts between strength and toughness. Nat Mater 10:817–822CrossRefPubMedCentralGoogle Scholar
  3. 3.
    Fratzl P, Weinkamer R (2007) Nature’s hierarchical materials. Prog Mater Sci 52:1263–1334CrossRefGoogle Scholar
  4. 4.
    Olszta MJ, Cheng XG, Jee SS, Kumar R, Kim YY, Kaufman MJ, Douglas EP, Gower LB (2007) Bone structure and formation: A new perspective. Mater Sci Eng R 58:77–116CrossRefGoogle Scholar
  5. 5.
    Goldberg M, Boskey AL (1996) Lipids and biomineralizations. Prog Histochem Cyto 31:III1–I187Google Scholar
  6. 6.
    Boskey AL (2001) Bone mineralization. In: Cowin SC (ed) Bone mechanics handbook. CRC Press, Boca Raton, pp 5.1–5.33Google Scholar
  7. 7.
    Baselt DR, Revel JP, Baldeschwieler JD (1993) Subfibrillar structure of type I collagen observed by atomic force microscopy. Biophys J 65:2644–2655CrossRefPubMedCentralGoogle Scholar
  8. 8.
    Fratzl P (2008) Collagen: structure and mechanics. Springer, New YorkCrossRefGoogle Scholar
  9. 9.
    Landis WJ, Song MJ, Leith A, McEwen L, McEwen BF (1993) Mineral and organic matrix interaction in normally calcifying tendon visualized in 3 dimensions by high-voltage electron-microscopic tomography and graphic image reconstruction. J Struct Biol 110:39–54CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Currey JD (2002) Bones: structure and mechanics. Princeton University Press, PrincetonGoogle Scholar
  11. 11.
    Barkaoui A, Bettamer A, Hambli R (2011) Failure of mineralized collagen microfibrils using finite element simulation coupled to mechanical quasi-brittle damage. Procedia Eng 10:3185–3190CrossRefGoogle Scholar
  12. 12.
    Barkaoui A, Bettamer A, Hambli R (2012) Mechanical behavior of single mineralized collagen fibril using finite element simulation coupled to quasi-brittle damage law. In: ECCOMAS, pp. 1357–1365Google Scholar
  13. 13.
    Barkaoui A, Hambli R (2014) Nanomechanical properties of mineralised collagen microfibrils based on finite elements method: biomechanical role of cross-links. Comput Methods Biomech Biomed Eng 17:1590–1601CrossRefGoogle Scholar
  14. 14.
    Barkaoui A, Hambli R, Tavares JMRS (2015) Effect of material and structural factors on fracture behaviour of mineralised collagen microfibril using finite element simulation. Comput Methods Biomech Biomed Eng 18:1181–1190CrossRefGoogle Scholar
  15. 15.
    Schwarcz HP (2015) The ultrastructure of bone as revealed in electron microscopy of ion-milled sections. Semin Cell Dev Biol 46:44–50CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Benezra Rosen V, Hobbs LW, Spector M (2002) The ultrastructure of anorganic bovine bone and selected synthetic hyroxyapatites used as bone graft substitute materials. Biomaterials 23:921–928CrossRefPubMedCentralGoogle Scholar
  17. 17.
    Chen P-Y, Toroian D, Price PA, McKittrick J (2011) Minerals form a continuum phase in mature cancellous bone. Calcif Tissue Int 81:351–361CrossRefGoogle Scholar
  18. 18.
    Chen P-Y, McKittrick J (2011) Compressive mechanical properties of demineralized and deproteinized cancellous bone. J Mech Behav Biomed 4:961–973CrossRefGoogle Scholar
  19. 19.
    Novitskaya E, Chen PY, Lee S, Castro-Cesena A, Hirata G, Lubarda VA, McKittrick J (2011) Anisotropy in the compressive mechanical properties of bovine cortical bone and the mineral and protein constituents. Acta Biomater 7:3170–3177CrossRefPubMedCentralGoogle Scholar
  20. 20.
    Bigi A, Ripamonti A, Cojazzi G, Pizzuto G, Roveri N, Koch MHJ (1991) Structural analysis of turkey tendon collagen upon removal of the inorganic phase. Int J Biol Macromol 13:110–114CrossRefPubMedCentralGoogle Scholar
  21. 21.
    Venkatesan J, Qian ZJ, Ryu B, Thomas NV, Kim SK (2011) A comparative study of thermal calcination and an alkaline hydrolysis method in the isolation of hydroxyapatite from Thunnus obesus bone. Biomed Mater 6:035003CrossRefPubMedCentralGoogle Scholar
  22. 22.
    Barakat NAM, Khalil KA, Sheikh FA, Omran AM, Gaihre B, Khil SM, Kim HY (2008) Physiochemical characterizations of hydroxyapatite extracted from bovine bones by three different methods: extraction of biologically desirable HAp. Mater Sci Eng C 28:1381–1387CrossRefGoogle Scholar
  23. 23.
    Barakat NAM, Khil MS, Omran AM, Sheikh FA, Kim HY (2009) Extraction of pure natural hydroxyapatite from the bovine bones bio waste by three different methods. J Mater Process Technol 209:3408–3415CrossRefGoogle Scholar
  24. 24.
    Ooi CY, Hamdi M, Ramesh S (2007) Properties of hydroxyapatite produced by annealing of bovine bone. Ceram Int 33:1171–1177CrossRefGoogle Scholar
  25. 25.
    Toque JA, Herliansyah MK, Hamdi M, Ide-Ektessabi A, Wildan MW (2007) The effect of sample preparation and calcination temperature on the production of hydroxyapatite from bovine bone powders. In: Ibrahim F, Osman NAA, Usman J, Kadri NA (eds) 3rd Kuala Lumpur International Conference on Biomedical Engineering 2006: Biomed 2006, 11–14 December 2006 Kuala Lumpur, Malaysia. Springer, Berlin, pp 152–155Google Scholar
  26. 26.
    Termine JD, Eanes ED, Greenfield DJ, Nylen MU, Harper RA (1973) Hydrazine-deproteinated bone mineral. Calcif Tissue Res 12:73–90CrossRefPubMedCentralGoogle Scholar
  27. 27.
    Bertazzo S, Bertran CA (2008) Effect of hydrazine deproteination on bone mineral phase: a critical view. J Inorg Biochem 102:137–145CrossRefPubMedCentralGoogle Scholar
  28. 28.
    Kim HM, Rey C, Glimcher MJ (1995) Isolation of calcium-phosphate crystals of bone by non-aqueous methods at low temperature. J Bone Miner Res 10:1589–1601CrossRefPubMedCentralGoogle Scholar
  29. 29.
    Tomazic BB, Brown WE, Eanes ED (1993) A critical evaluation of the purification of biominerals by hypochlorite treatment. J Biomed Mater Res A 27:217–225CrossRefGoogle Scholar
  30. 30.
    Wynnyckyj C, Omelon S, Willett T, Kyle K, Goldberg H, Grynpas M (2011) Mechanism of bone collagen degradation due to KOH treatment. Biochim. Biophys Acta Gen Subj 1810:192–201CrossRefGoogle Scholar
  31. 31.
    Karlsmark T, Danielsen L, Thomsen HK, Aalund O, Nielsen KG, Nielsen O, Genefke IK (1988) The effect of sodium hydroxide and hydrochloric acid on pig dermis. A light microscopic study. Forensic Sci Int 39:227–233CrossRefPubMedCentralGoogle Scholar
  32. 32.
    Uklejewski R, Winiecki M, Musielak G, Tokłowicz R (2015) Effectiveness of various deproteinization processes of bovine cancellous bone evaluated via mechano-biostructural properties of produced osteoconductive biomaterials. Biotechnol Bioprocess E 20:259–266CrossRefGoogle Scholar
  33. 33.
    Weiner S, Price PA (1986) Disaggregation of bone into crystals. Calcif Tissue Int 39:365–375CrossRefPubMedCentralGoogle Scholar
  34. 34.
    Chen P-Y, Toroian D, Price PA, McKittrick J (2011) Minerals form a continuum phase in mature cancellous bone. Calcif Tissue Int 88:351–361CrossRefPubMedCentralGoogle Scholar
  35. 35.
    Termine JD, Belcourt AB, Conn KM, Kleinman HK (1981) Mineral and collagen-binding proteins of fetal calf bone. J Biol Chem 256:403–408Google Scholar
  36. 36.
    Termine JD, Conn KM, Kleinman HK, Martin GR, Whitson SW (1981) Osteonectin, a mineral and collagen binding-protein of fetal calf bone. Calcif Tissue Int 33:302–302Google Scholar
  37. 37.
    Glimcher MJ (1989) Mechanism of calcification: role of collagen fibrils and collagen phosphoprotein complexes in vitro and in vivo. Anat Rec 224:139–153CrossRefPubMedCentralGoogle Scholar
  38. 38.
    Morris MD, Mandair GS (2011) Raman assessment of bone quality. Clin Orthop Relat R 469:2160–2169CrossRefGoogle Scholar
  39. 39.
    Mandair GS, Morris MD (2015) Contributions of Raman spectroscopy to the understanding of bone strength. BoneKEy Rep 4:620CrossRefPubMedCentralGoogle Scholar
  40. 40.
    Akkus O, Adar F, Schaffler MB (2004) Age-related changes in physicochemical properties of mineral crystals are related to impaired mechanical function of cortical bone. Bone 34:443–453CrossRefPubMedCentralGoogle Scholar
  41. 41.
    Martiniaková M, Grosskopf B, Omelka R, Vondrakova M, Bauerova M (2006) Differences among species in compact bone tissue microstructure of mammalian skeleton: use of a discriminant function analysis for species identification. J Forensic Sci 51:1235–1239CrossRefPubMedCentralGoogle Scholar
  42. 42.
    Urist MR, Behnam K, Kerendi F, Raskin K, Nuygen TD, Shamie AN, Malinin TI (1997) Lipids closely associated with bone morphogenetic protein (BMP) and induced heterotopic bone formation. With preliminary observations of deficiencies in lipid and osteoinduction in lathyrism in rats. Connect Tissue Res 36:9–20CrossRefPubMedCentralGoogle Scholar
  43. 43.
    Fages J, Marty A, Delga C, Condoret JS, Combes D, Frayssinet P (1994) Use of supercritical CO2 for bone delipidation. Biomaterials 15:650–656CrossRefPubMedCentralGoogle Scholar
  44. 44.
    Feng L, Jasiuk I (2011) Multi-scale characterization of swine femoral cortical bone. J Biomech 44:313–320CrossRefPubMedCentralGoogle Scholar
  45. 45.
    Locke M (2004) Structure of long bones in mammals. J Morphol 262:546–565CrossRefPubMedCentralGoogle Scholar
  46. 46.
    Almany Magal R, Reznikov N, Shahar R, Weiner S (2014) Three-dimensional structure of minipig fibrolamellar bone: adaptation to axial loading. J Struct Biol 186:253–264CrossRefPubMedCentralGoogle Scholar
  47. 47.
    Chittenden M, Najafi AR, Li J, Jasiuk I (2015) Nanoindentation and ash content study of age dependent changes in porcine cortical bone. J Mech Med Biol 15:1550074CrossRefGoogle Scholar
  48. 48.
    Martiniaková M, Grosskopf B, Omelka R, Dammers K, Vondráková M, Bauerová M (2007) Histological study of compact bone tissue in some mammals: a method for species determination. Int J Osteoarchaeol 17:82–90CrossRefGoogle Scholar
  49. 49.
    Mehdawi IM, Young A (2015) Antibacterial composite restorative materials for dental applications. In: Biomaterials and medical device—associated infections. Woodhead Publishing, Oxford, pp 199–221CrossRefGoogle Scholar
  50. 50.
    Bagambisa F, Joos U, Schilli W (1993) A scanning electron microscope study of the ultrastructural organization of bone mineral. Cell Mater 3:10Google Scholar
  51. 51.
    Chen P-Y, Novitskaya E, Sun C-Y, McKittrick J, Lopez MI (2014) Toward a better understanding of mineral microstructure in bony tissues. Bioinspired Biomimetic Nanobiomaterials 3:71–84CrossRefGoogle Scholar
  52. 52.
    Fratzl P, Schreiber S, Klaushofer K (1996) Bone mineralization as studied by small-angle X-ray scattering. Connect Tissue Res 35:9–16Google Scholar
  53. 53.
    Hamed E, Novitskaya EE, Li J, Chen P-Y, Jasiuk I, McKittrick J (2012) Elastic moduli of untreated, demineralized, and deproteinized cortical bone: validation of a theoretical model of bone as an interpenetrating composite material. Acta Biomater 8:1080–1092CrossRefPubMedCentralGoogle Scholar
  54. 54.
    Di Renzo M, Ellis TH, Sacher E, Stangel I (2001) A photoacoustic FTIRS study of the chemical modifications of human dentin surfaces: II. Deproteination Biomater 22:793–797CrossRefGoogle Scholar
  55. 55.
    Tas AC (2012) X-ray diffraction data for flux-grown calcium hydroxyapatite whiskers. Powder Diffr 16:102–106CrossRefGoogle Scholar
  56. 56.
    Jackson SA, Cartwright AG, Lewis D (1978) The morphology of bone mineral crystals. Calcif Tissue Res 25:217–222CrossRefPubMedCentralGoogle Scholar
  57. 57.
    Meneghini C, Dalconi MC, Nuzzo S, Mobilio S, Wenk RH (2003) Rietveld refinement on X-ray diffraction patterns of bioapatite in human fetal bones. Biophys J 84:2021–2029CrossRefPubMedCentralGoogle Scholar
  58. 58.
    Rey C, Shimizu M, Collins B, Glimcher MJ (1990) Resolution-enhanced fourier transform infrared spectroscopy study of the environment of phosphate ions in the early deposits of a solid phase of calcium-phosphate in bone and enamel, and their evolution with age. I: investigations in the v4 PO4 domain. Calcif Tissue Int 46:384–394CrossRefPubMedCentralGoogle Scholar
  59. 59.
    Francis MD, Webb NC (1970) Hydroxyapatite formation from a hydrated calcium monohydrogen phosphate precursor. Calcif Tissue Int 6:335–342CrossRefGoogle Scholar
  60. 60.
    Lin F-H, Lin C-C, Lu C-M, Liu H-C, Sun J-S, Wang C-Y (1995) Mechanical properties and histological evaluation of sintered β-Ca2P2O7 with Na4P2O7 · 10H2O addition. Biomaterials 16:793–802CrossRefPubMedCentralGoogle Scholar
  61. 61.
    Johnsson MS-A, Nancollas GH (1992) The role of brushite and octacalcium phosphate in apatite formation. Crit Rev Oral Biol Med 3:61–82CrossRefPubMedCentralGoogle Scholar
  62. 62.
    Crane NJ, Popescu V, Morris MD, Steenhuis P, Ignelzi MA (2006) Raman spectroscopic evidence for octacalcium phosphate and other transient mineral species deposited during intramembranous mineralization. Bone 39:434–442CrossRefPubMedCentralGoogle Scholar
  63. 63.
    Czamara K, Majzner K, Pacia MZ, Kochan K, Kaczor A, Baranska M (2015) Raman spectroscopy of lipids: a review. J Raman Spectrosc 46:4–20CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Frances Y. Su
    • 1
  • Siyuan Pang
    • 2
  • Yik Tung Tracy Ling
    • 2
  • Peter Shyu
    • 2
  • Ekaterina Novitskaya
    • 3
  • Kyungah Seo
    • 1
  • Sofia Lambert
    • 4
  • Kimberlin Zarate
    • 5
  • Olivia A. Graeve
    • 3
  • Iwona Jasiuk
    • 2
    • 6
  • Joanna McKittrick
    • 1
    • 3
    • 7
  1. 1.Materials Science and Engineering ProgramUniversity of California, San DiegoLa JollaUSA
  2. 2.Department of Mechanical Science and EngineeringUniversity of Illinois at Urbana ChampaignUrbanaUSA
  3. 3.Department of Mechanical and Aerospace EngineeringUniversity of California, San DiegoLa JollaUSA
  4. 4.Centro de Enseñanza Técnica y Superior - Campus MexicaliMexicaliMexico
  5. 5.Hilltop High SchoolChula VistaUSA
  6. 6.University of Illinois at Urbana-ChampaignUrbanaUSA
  7. 7.University of California, San DiegoLa JollaUSA

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