Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress 2023 EHA Congress 2023 ASCO Annual Meeting
Clinical Collections
Advances in Alzheimer’s

At a glance: The ONWARDS insulin icodec trials

A round-up of the ONWARDS phase 3 clinical trials evaluating the novel weekly insulin icodec in people with diabetes.
ADVANCED SEARCH
Log in
Top
Calcified Tissue International
Published in: Calcified Tissue International 5/2018

01-11-2018 | Original Research

Deproteinization of Cortical Bone: Effects of Different Treatments

Authors: 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

Published in: Calcified Tissue International | Issue 5/2018

Login to get access

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.
Appendix
Available only for authorised users
Literature
1.
Weiner S, Wagner HD (1998) The material bone: Structure mechanical function relations. Annu Rev Mater Sci 28:271–298CrossRef
2.
3.
Fratzl P, Weinkamer R (2007) Nature’s hierarchical materials. Prog Mater Sci 52:1263–1334CrossRef
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 58:77–116CrossRef
5.
Goldberg M, Boskey AL (1996) Lipids and biomineralizations. Prog Histochem Cyto 31:III1–I187
6.
Boskey AL (2001) Bone mineralization. In: Cowin SC (ed) Bone mechanics handbook. CRC Press, Boca Raton, pp 5.1–5.33
7.
Baselt DR, Revel JP, Baldeschwieler JD (1993) Subfibrillar structure of type I collagen observed by atomic force microscopy. Biophys J 65:2644–2655CrossRefPubMedCentral
8.
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 110:39–54CrossRefPubMedCentral
10.
Currey JD (2002) Bones: structure and mechanics. Princeton University Press, Princeton
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 10:3185–3190CrossRef
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. 1357–1365
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 17:1590–1601CrossRef
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 18:1181–1190CrossRef
15.
Schwarcz HP (2015) The ultrastructure of bone as revealed in electron microscopy of ion-milled sections. Semin Cell Dev Biol 46:44–50CrossRefPubMedCentral
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 23:921–928CrossRefPubMedCentral
17.
Chen P-Y, Toroian D, Price PA, McKittrick J (2011) Minerals form a continuum phase in mature cancellous bone. Calcif Tissue Int 81:351–361CrossRef
18.
Chen P-Y, McKittrick J (2011) Compressive mechanical properties of demineralized and deproteinized cancellous bone. J Mech Behav Biomed 4:961–973CrossRef
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 7:3170–3177CrossRefPubMedCentral
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 13:110–114CrossRefPubMedCentral
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 6:035003CrossRefPubMedCentral
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 28:1381–1387CrossRef
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 209:3408–3415CrossRef
24.
Ooi CY, Hamdi M, Ramesh S (2007) Properties of hydroxyapatite produced by annealing of bovine bone. Ceram Int 33:1171–1177CrossRef
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 152–155
26.
Termine JD, Eanes ED, Greenfield DJ, Nylen MU, Harper RA (1973) Hydrazine-deproteinated bone mineral. Calcif Tissue Res 12:73–90CrossRefPubMedCentral
27.
Bertazzo S, Bertran CA (2008) Effect of hydrazine deproteination on bone mineral phase: a critical view. J Inorg Biochem 102:137–145CrossRefPubMedCentral
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 10:1589–1601CrossRefPubMedCentral
29.
Tomazic BB, Brown WE, Eanes ED (1993) A critical evaluation of the purification of biominerals by hypochlorite treatment. J Biomed Mater Res A 27:217–225CrossRef
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 1810:192–201CrossRef
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 39:227–233CrossRefPubMedCentral
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 20:259–266CrossRef
33.
34.
Chen P-Y, Toroian D, Price PA, McKittrick J (2011) Minerals form a continuum phase in mature cancellous bone. Calcif Tissue Int 88:351–361CrossRefPubMedCentral
35.
Termine JD, Belcourt AB, Conn KM, Kleinman HK (1981) Mineral and collagen-binding proteins of fetal calf bone. J Biol Chem 256:403–408
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 33:302–302
37.
Glimcher MJ (1989) Mechanism of calcification: role of collagen fibrils and collagen phosphoprotein complexes in vitro and in vivo. Anat Rec 224:139–153CrossRefPubMedCentral
38.
Morris MD, Mandair GS (2011) Raman assessment of bone quality. Clin Orthop Relat R 469:2160–2169CrossRef
39.
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 34:443–453CrossRefPubMedCentral
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 51:1235–1239CrossRefPubMedCentral
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 36:9–20CrossRefPubMedCentral
43.
Fages J, Marty A, Delga C, Condoret JS, Combes D, Frayssinet P (1994) Use of supercritical CO2 for bone delipidation. Biomaterials 15:650–656CrossRefPubMedCentral
44.
45.
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 186:253–264CrossRefPubMedCentral
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 15:1550074CrossRef
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 17:82–90CrossRef
49.
Mehdawi IM, Young A (2015) Antibacterial composite restorative materials for dental applications. In: Biomaterials and medical device—associated infections. Woodhead Publishing, Oxford, pp 199–221CrossRef
50.
Bagambisa F, Joos U, Schilli W (1993) A scanning electron microscope study of the ultrastructural organization of bone mineral. Cell Mater 3:10
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 3:71–84CrossRef
52.
Fratzl P, Schreiber S, Klaushofer K (1996) Bone mineralization as studied by small-angle X-ray scattering. Connect Tissue Res 35:9–16
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 8:1080–1092CrossRefPubMedCentral
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 22:793–797CrossRef
55.
Tas AC (2012) X-ray diffraction data for flux-grown calcium hydroxyapatite whiskers. Powder Diffr 16:102–106CrossRef
56.
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 84:2021–2029CrossRefPubMedCentral
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 46:384–394CrossRefPubMedCentral
59.
Francis MD, Webb NC (1970) Hydroxyapatite formation from a hydrated calcium monohydrogen phosphate precursor. Calcif Tissue Int 6:335–342CrossRef
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 16:793–802CrossRefPubMedCentral
61.
Johnsson MS-A, Nancollas GH (1992) The role of brushite and octacalcium phosphate in apatite formation. Crit Rev Oral Biol Med 3:61–82CrossRefPubMedCentral
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 39:434–442CrossRefPubMedCentral
63.
Czamara K, Majzner K, Pacia MZ, Kochan K, Kaczor A, Baranska M (2015) Raman spectroscopy of lipids: a review. J Raman Spectrosc 46:4–20CrossRef
Metadata
Title
Deproteinization of Cortical Bone: Effects of Different Treatments
Authors
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
Publication date
01-11-2018
Publisher
Springer US
Published in
Calcified Tissue International / Issue 5/2018
Print ISSN: 0171-967X
Electronic ISSN: 1432-0827
DOI
https://doi.org/10.1007/s00223-018-0453-x

Other articles of this Issue 5/2018

Calcified Tissue International 5/2018 Go to the issue

Original Research

Abaloparatide is an Effective Treatment Option for Postmenopausal Osteoporosis: Review of the Number Needed to Treat Compared with Teriparatide

Original Research

Longitudinal Change in Peripheral Quantitative Computed Tomography Assessment in Older Adults: The Hertfordshire Cohort Study

Original Research

Diet Quality and Bone Measurements Using HRpQCT and pQCT in Older Community-Dwelling Adults from the Hertfordshire Cohort Study

Original Research

The C-Terminal Intact Forms of Periostin (iPTN) Are Surrogate Markers for Osteolytic Lesions in Experimental Breast Cancer Bone Metastasis

Original Research

Is There Causal Relationship of Smoking and Alcohol Consumption with Bone Mineral Density? A Mendelian Randomization Study

Original Research

Comparison of Bone Microarchitecture Between Adult Osteogenesis Imperfecta and Early-Onset Osteoporosis

ACC 2024 Congress Coverage

Heart Failure Video

Year in Review: Heart failure and cardiomyopathies

Watch now

Watch this official video from ACC.24. Dr. Biykem Bozkurt discuss last year's major advances in heart failure and cardiomyopathies.

Semaglutide benefits people with type 2 diabetes and obesity-related heart failure

16-04-2024 Type 2 Diabetes News

Incentivised to exercise

11-04-2024 Lifestyle Modifications News

New intervention for STEMI-related cardiogenic shock

10-04-2024 Myocardial Infarction News

» View more highlights on the ACC 2024 congress hub page

Image Credits