The Effect of Formalin Preservation Time and Temperature on the Material Properties of Bovine Femoral Cortical Bone Tissue
Literature has reported controversial findings on whether formalin affected bone properties, or not, especially when different preservation time durations and temperatures were involved. Hence, accurately and systematically quantifying the effect of formalin on the mechanical properties of bone using a large dataset is crucial for assessing biomechanical responses based on fixed specimens. A total of 154 longitudinal and 149 transverse cuboid-shaped (12 mm × 2 mm × 0.5 mm) specimens from the midsection of 12 bovine femora from six bovines were prepared and assigned to ten groups, including fresh-frozen, formalin-preserved at 25 °C for 4 weeks and 8 weeks, and formalin-preserved at 4 °C for 4 weeks and 8 weeks. All specimens underwent quasi-static three-point bending tests with a loading rate of 0.02 mm/s. The Young’s modulus, yield stress, yield strain, tangent modulus, effective plastic strain, ultimate stress, and toughness were calculated by optimizing the material parameters to make the force–displacement curve of the finite element prediction consistent with the experimental curve, combined with specimen-specific finite element models. Preservation time and temperature both had significant effects on the Young’s modulus, yield stress, effective plastic strain, yield strain and ultimate stress of cortical bone (p < 0.05). The Young’s modulus, yield stress, and ultimate stress of longitudinal specimens decreased significantly with the increase of preservation time, and the yield strain increased significantly. As the preservation temperature increases, the Young’s modulus of the transverse sample decreased significantly, and the yield strain increased significantly. The preservation time mainly affects the longitudinal specimens, while the preservation temperature mainly affects the transverse specimens. Formalin preservation of bovine femoral cortical bones at a lower temperature and less than 4 weeks is recommended for biomechanical testing.
KeywordsCortical bone Formalin fixation Mechanical properties Finite element method Optimization
This research was funded by the National Natural Science Foundation of China (51205118 and 11402296) and the State Key Laboratory of Advanced Design and Manufacturing for Vehicle Body, Hunan University (51475002). We also acknowledge support from the Canada Research Chairs program.
Conflict of interest
There are no conflicts of interest.
- 5.Baum, T., E. Grande Garcia, R. Burgkart, O. Gordijenko, H. Liebl, P. M. Jungmann, M. Gruber, T. Zahel, E. J. Rummeny, S. Waldt, and J. S. Bauer. Osteoporosis imaging: Effects of bone preservation on mdct-based trabecular bone microstructure parameters and finite element models. BMC Med. Imaging 15:22, 2015.CrossRefGoogle Scholar
- 6.Bian, D., J. Deng, N. Li, X. Chu, Y. Liu, W. Li, H. Cai, P. Xiu, Y. Zhang, Z. Guan, Y. Zheng, Y. Kou, B. Jiang, and R. Chen. In vitro and in vivo studies on biomedical magnesium low-alloying with elements gadolinium and zinc for orthopedic implant applications. ACS Appl. Mater. Interfaces 10:4394–4408, 2018.CrossRefGoogle Scholar
- 9.Cowin, S. C. Bone Mechanics Handbook (2nd ed.). Boca Raton, FL: CRC Press, 2001.Google Scholar
- 17.Healing, T. D., P. N. Hoffman, and S. E. Young. The infection hazards of human cadavers. Commun. Dis. Rep. CDR Rev. 5:R61–68, 1995.Google Scholar
- 31.Tong, J. Effects of fixative solution ph and Ca2+ concentration on decalcification of ancient corpse bone. Jianghan Archaeol. 106:113–116, 2008.Google Scholar
- 34.Turner, C. H., and D. B. Burr. Experimental techniques for bone mechanics. In: Bone Mechanics Handbook2nd, edited by S. C. Cowin. Boca Raton, FL: CPC Press, 2001, pp. 7–20.Google Scholar
- 37.Wieding, J., E. Mick, A. Wree, and R. Bader. Influence of three different preservative techniques on the mechanical properties of the ovine cortical bone. Acta Bioeng. Biomech. 17:137–146, 2015.Google Scholar
- 38.Wittenburg, G., C. Volkel, R. Mai, and G. Lauer. Immunohistochemical comparison of differentiation markers on paraffin and plastic embedded human bone samples. J. Physiol. Pharmacol. 60:43–49, 2009.Google Scholar
- 41.Zhang, G., J. Yang, F. Guan, D. Chen, N. Li, L. Cao, and H. Mao. Quantifying the effects of formalin fixation on the mechanical properties of cortical bone using beam theory and optimization methodology with specimen-specific finite element models. J. Biomech. Eng. 138:094502, 2016.CrossRefGoogle Scholar
- 42.Zimmermann, E. A., E. Schaible, B. Gludovatz, F. N. Schmidt, C. Riedel, M. Krause, E. Vettorazzi, C. Acevedo, M. Hahn, K. Puschel, S. Tang, M. Amling, R. O. Ritchie, and B. Busse. Intrinsic mechanical behavior of femoral cortical bone in young, osteoporotic and bisphosphonate-treated individuals in low- and high energy fracture conditions. Sci. Rep. 6:21072, 2016.CrossRefGoogle Scholar