Comparison of different protocols for demineralization of cortical bone

Pang, Siyuan; Su, Frances Y.; Green, Amesha; Salim, Justin; McKittrick, Joanna; Jasiuk, Iwona

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Pang, Siyuan¹; Su, Frances Y.²; Green, Amesha³; Salim, Justin⁴; McKittrick, Joanna²; Jasiuk, Iwona¹

Comparison of different protocols for demineralization of cortical bone

Sci Rep. March 29, 2021;11:7012

Affiliations

¹Department of Mechanical Science and Engineering, University of Illinois at Urbana Champaign, 1206 West Green Street, Urbana, IL 61801, USA

²Department of Mechanical and Aerospace Engineering and Materials Science and Engineering Program, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093‐0411, USA

³Department of Chemical, Biological, and Bio Engineering, North Carolina Agricultural and Technical State University, 1601 E Market St, Greensboro, NC 27401, USA

⁴Department of Bioengineering, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093‐0412, USA
Abstract

Bone is a biological composite material consisting of two main components: collagen and mineral. Collagen is the most abundant protein in vertebrates, which makes it of high clinical and scientific interest. In this paper, we compare the composition and structure of cortical bone demineralized using several protocols: ethylene-diamine-tetraacetic acid (EDTA), formic acid (CH₂O₂), hydrochloric acid (HCl), and HCl/EDTA mixture. The efficiencies of these four agents were investigated by assessing the remaining mineral quantities and collagen integrity with various experimental techniques. Raman spectroscopy results show that the bone demineralized by the CH₂O₂ agent has highest collagen quality parameter. The HCl/EDTA mixture removes the most mineral, but it affects the collagen secondary structure as amide II bands are shifted as observed by Fourier transform infrared spectroscopy. Thermogravimetric analysis reveals that HCl and EDTA are most effective in removing the mineral with bulk measurements. In summary, we conclude that HCl best demineralizes bone, leaving the well-preserved collagen structure in the shortest time. These findings guide on the best demineralization protocol to obtain high-quality collagen from bone for clinical and scientific applications.
Links

DOI: 10.1038/s41598-021-86257-4
History

Received: 2020-10-10

Accepted: 2021-03-03

Online: 2021-03-29

Cited Works (12)

Cited By (1)

Year	Entry
1992	Weiner S, Traub W. Bone structure: from ȧngstroms to microns. FASEB J. February 1992;6(3):879-885.
1996	Gadaleta SJ, Paschalis EP, Betts F, Mendelsohn R, Boskey AL. Fourier transform infrared spectroscopy of the solution-mediated conversion of amorphous calcium phosphate to hydroxyapatite: new correlations between X-ray diffraction and infrared data. Calcif Tiss Int. January 1996;58(1):9-16.
1998	Rho J-Y, Kuhn-Spearing L, Zioupos P. Mechanical properties and the hierarchical structure of bone. Med Eng Phys. 1998;20(2):92-102.
2002	Wang X, Shen X, Li X, Agrawal CM. Age-related changes in the collagen network and toughness of bone [published correction appears in Bone. 2003;32(1):107]. Bone. 2002;31(1):1-7.
2002	Currey JD. Bones: Structure and Mechanics. Princeton, NJ: Princeton University Press; 2002.
2006	Yerramshetty JS, Lind C, Akkus O. The compositional and physicochemical homogeneity of male femoral cortex increases after the sixth decade. Bone. December 2006;39(6):1236-1243.
2007	Olszta MJ, Cheng X, Jee SS, Kumar R, Kim Y-Y, Kaufman MJ, Douglas EP, Gower LB. Bone structure and formation: a new perspective. Mater Sci Eng R Rep. November 28, 2007;58(3-5):77-116.
2008	Yerramshetty JS, Akkus O. The associations between mineral crystallinity and the mechanical properties of human cortical bone. Bone. March 2008;42(3):476-482.
2011	Morris MD, Mandair GS. Raman assessment of bone quality. Clin Orthop Relat Res. August 2011;469:2160-2169.
2011	Chen P-Y, Toroian D, Price PA, McKittrick J. Minerals form a continuum phase in mature cancellous bone. Calcif Tiss Int. May 2011;88(5):351-361.
2011	Chen P-Y, McKittrick J. Compressive mechanical properties of demineralized and deproteinized cancellous bone. J Mech Behav Biomed Mater. October 2011;4(7):961-973.
2015	Mandair GS, Morris MD. Contributions of Raman spectroscopy to the understanding of bone strength. BoneKEy Rep. January 7, 2015;4:620.