Deproteinization of Cortical Bone: Effects of Different Treatments
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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.
KeywordsDeproteinization Thermogravimetric analysis Cortical bone Raman spectroscopy Scanning electron microscopy
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.
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