Nanoindentation of Bio- and Geo-Mineralized Composites: Contribution of Microstructure and Composition

Bio-mineralized composite tissues, such as bone and teeth, are heterogeneous in both mineral composition and crystallinity. These tissues are altered throughout life by aging, wear, microfracture, and various disease states (e.g., osteoporosis), and are then further altered geologically after death by fossilization to create geo-minealized tissues. Bone and enamel exhibit a wide range of mechanical responses at nanometer-length scales, where large-scale porosity and macro-structural variation are not factors. Some variability seen in mineralized tissues can be attributed to the amount of mineral; where a general increase in mechanical properties occurs with increasing mineral volume fraction. However, a large range of modulus values for bone is observed at a constant mineral content indicating that both the composition and microstructure play a vital role in the nanomechanical response. This dissertation is aimed at understanding the nanomechanical properties of these heterogeneous mineralized composites in order to elucidate the interplay between composition, microstructure, and tissue mechanical behavior.

A combined approach using nanoindentation testing and complimentary techniques, such as X-ray diffraction, Fourier transform infrared spectroscopy, and quantitative backscattered electron microscopy (qBSE) are used to investigate the effect of variations in crystallography, microstructure, and mineral composition on the nanomechanical properties of these materials. Further, development of novel qBSE glass standards allow for site-matched measurement of mineral volume fraction and nanomechanical properties. Additionally, a finite element analysis (FEA) allows for isolation of individual parameters and their contribution to the nanomechanical properties. The relative contribution of the composition and microstructure are explored through two experimental model systems of bone fossilization and lemur enamel.

In fossilization, or diagenesis, composition is altered over geologic time as minerals are incorporated into pore spaces within the bone. Fossilized bone samples demonstrate a larger range of mineral composition, mineral volume fraction, and crystallinity than is found in modern samples. Nanoindentation revealed that anisotropy of modern bone can be preserved in fossil bones going back at least to the early Eocene (≈ 50 million years). Further, both increased crystallinity and density correlated with increased modulus values, suggesting that size of bioapatite crystals contribute to the mechanical properties. Nanoindentation is useful in investigating tissue-level diagenesis in bone, and can provide insight into the functional significance of mineralized tissues even after diagenesis has occurred.

Variations in microstructure and mineralization were examined in the enamel of three lemur species Lemur catta, Lepilemur leucopus, Propithecus verreauxi, and Homo sapiens. Nanoindentation revealed a natural gradation of mechanical properties where a 2-12% increase in modulus and hardness correlated to increased mineral content (p <0.001) measured by qBSE. Enamel microcracking in Lemur catta resulted in a 49% reduction in nanomechcanical properties at the occlusal (or chewing) surface of the tooth. FEA modeling demonstrated a similar decrease in modulus values for indentation within 20 microns of a crack. Variations in enamel microstructure and microcracking in lemur species enables study of the interplay between tissue microstructure and nanomechanical properties, and further explores variations with diet.

The investigation of nanomechanical property dependence on microstructure and mineral composition in two experimental model systems combined with FEA is used to understand the fundamental mechanical behavior of biological heterogeneous composite materials. Understanding the interplay between material structure and function in biomineralized composites will help to elucidate the relative contributions of various factors to nanomechanical behavior and will ultimately lead to improved development of biomimetic materials.

Year	Entry
1994	Mente PL, Lewis JL. Elastic modulus of calcified cartilage is an order of magnitude less than that of subchondral bone. J Orthop Res. September 1994;12(5):637-647.
1984	Lees S, Bonar LC, Mook HA. A study of dense mineralized tissue by neutron diffraction. Int J Biol Macromol. 1984;6(6):321-326.
2000	Boivin GY, Chavassieux PM, Santora AC, Yates J, Meunier PJ. Alendronate increases bone strength by increasing the mean degree of mineralization of bone tissue in osteoporotic women. Bone. November 2000;27(5):687-694.
2007	Lievers WB, Lee V, Arsenault SM, Waldman SD, Pilkey AK. Specimen size effect in the volumetric shrinkage of cancellous bone measured at two levels of dehydration. J Biomech. 2007;40(9):1903-1909.
1996	Landis WJ, Hodgens KJ, Arena J, Song MJ, McEwen BF. Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography. Microsc Res Tech. 1996;33(2):192-202.
1967	Ascenzi A, Bonucci E. The tensile properties of single osteons. Acta Anat. 1967;158(4):375-386.
1963	Hashin Z, Shtrikman S. A variational approach to the theory of the elastic behaviour of multiphase materials. J Mech Phys Solids. March–April 1963;11(2):127-140.
2002	Fan Z, Swadener JG, Rho JY, Roy ME, Pharr GM. Anisotropic properties of human tibial cortical bone as measured by nanoindentation. J Orthop Res. July 2002;20(4):806-810.
2003	Fan Z, Rho J-Y. Effects of viscoelasticity and time-dependent plasticity on nanoindentation measurements of human cortical bone. J Biomed Mater Res. October 1, 2003;A67(1):208-214.
1988	Ascenzi A. The micromechanics versus the macromechanics of cortical bone: a comprehensive presentation. J Biomech Eng. November 1988;110(4):357-363.
2006	Hofmann T, Heyroth F, Meinhard H, Fränzel W, Raum K. Assessment of composition and anisotropic elastic properties of secondary osteon lamellae. J Biomech. 2006;39(12):2282-2294.
1964	Gong JK, Arnold JS, Cohn SH. Composition of trabecular and cortical bone. Anat Rec. 1964;149(3):325-331.
1951	Evans FG, Lebow M. Regional differences in some of the physical properties of the human femur. J Appl Physiol. 1951;3(9):563-572.
1958	Amprino R. Investigations on some physical properties of bone tissue. Acta Anat. 1958;34(3):161-186.
1993	Skedros JG, Bloebaum RD, Bachus KN, Boyce TM. The meaning of graylevels in backscattered electron images of bone. J Biomed Mater Res. January 1993;7(1):47-56.
2003	Ferguson VL, Bushby AJ, Boyde A. Nanomechanical properties and mineral concentration in articular calcified cartilage and subchondral bone. J Anat. August 2003;203(2):191-202.
2000	Carden A, Morris MD. Application of vibrational spectroscopy to the study of mineralized tissues (review). J Biomed Opt. July 2000;5(3):259-268.
1993	Boyde A, Elliott JC, Jones SJ. Stereology and histogram analysis of backscattered electron images: age changes in bone. Bone. May–June 1993;14(3):205-210.
1996	Pidaparti RMV, Chandran A, Takano Y, Turner CH. Bone mineral lies mainly outside collagen fibrils: predictions of a composite model for osternal bone. J Biomech. July 1996;29(7):909-916.
1992	Oliver WC, Pharr GM. An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J Mater Res. 1992;7(6):1564-1583.
1999	Boyde A, Travers R, Glorieux FH, Jones SJ. The mineralization density of iliac crest bone from children with osteogenesis imperfecta. Calcif Tiss Int. March 1999;64(3):185-190.
2000	Jäger I, Fratzl P. Mineralized collagen fibrils: a mechanical model with a staggered arrangement of mineral particles. Biophys J. October 2000;79(4):1737-1746.
1995	Sasaki N, Enyo A. Viscoelastic properties of bone as a function of water content. J Biomech. 1995;28(7):809-815.
2006	Barthelat F, Li C-M, Comi C, Espinosa HD. Mechanical properties of nacre constituents and their impact on mechanical performance. J Mater Sci. 2006;21(8):1977-1986.
2002	Hengsberger S, Kulik A, Zysset P. Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. Bone. 2002;30(1):178-184.
2007	Ramasamy JG, Akkus O. Local variations in the micromechanical properties of mouse femur: the involvement of collagen fiber orientation and mineralization. J Biomech. 2007;40(4):910-918.
2000	Hoffler CE, Moore KE, Kozloff K, Zysset PK, Brown MB, Goldstein SA. Heterogeneity of bone lamellar-level elastic moduli. Bone. June 2000;26(6):603-609.
2004	Oliver WC, Pharr GM. Measurement of hardness and elastic modulus by instrumented indentation: advances in understanding and refinements to methodology. J Mater Res. January 2004;19(1):3-20.
1994	Finlay JB, Hardie WR. Anisotropic contraction of cortical bone caused by dehydration of samples of the bovine femur in vitro. Proc Inst Mech Eng Part H-J Eng Med. 1994;208(1):27-32.
2003	Burr DB, Miller L, Grynpas M, Li J, Boyde A, Mashiba T, Hirano T, Johnston CC. Tissue mineralization is increased following 1-year treatment with high doses of bisphosphonates in dogs. Bone. December 2003;33(6):960-969.
2006	Bembey AK, Bushby AJ, Boyde A, Ferguson VL, Oyen ML. Hydration effects on the micro-mechanical properties of bone. J Mater Res. August 2006;21(8):1962-1968.
1998	Zysset PK, Guo XE, Hoffler CE, Moore KE, Goldstein SA. Mechanical properties of human trabecular bone lamellae quantified by nanoindentation. Technol Health Care. December 1998;6(5-6):429-432.
1999	Zysset PK, Guo XE, Hoffler CE, Moore KE, Goldstein SA. Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur. J Biomech. October 1999;32(10):1005-1012.
1997	Bloebaum RD, Skedros JG, Vajda EG, Bachus KN, Constantz BR. Determining mineral content variations in bone using backscattered electron imaging. Bone. May 1997;20(5):485-490.
2010	Zhang J, Michalenko MM, Kuhl E, Ovaert TC. Characterization of indentation response and stiffness reduction of bone using a continuum damage model. J Mech Behav Biomed Mater. February 2010;3(2):189-202.
2001	Rho JY, Currey JD, Zioupos P, Pharr GM. The anisotropic Young's modulus of equine secondary osteones and interstitial bone determined by nanoindentation. J Exp Biol. May 2001;204(10):1775-1781.
2002	Rho JY, Zioupos P, Currey JD, Pharr GM. Microstructural elasticity and regional heterogeneity in human femoral bone of various ages examined by nano-indentation. J Biomech. February 2002;35(2):189-198.
1991	Lee DD, Glimcher MJ. Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone. J Mol Biol. 1991;217(3):487-501.
2006	Kazanci M, Roschger P, Paschalis EP, Klaushofer K, Fratzl P. Bone osteonal tissues by Raman spectral mapping: orientation–composition. J Struct Biol. December 2006;156(3):489-496.
1964	Currey JD. Three analogies to explain the mechanical properties of bone. Biorheology. 1964;2(1):1-10.
1999	Currey JD. The design of mineralised hard tissues for their mechanical functions. J Exp Biol. December 1999;202(23):3285-3294.
1999	Rho JY, Zioupos P, Currey JD, Pharr GM. Variations in the individual thick lamellar properties within osteons by nanoindentation. Bone. September 1999;25(3):295-300.
1999	Rho J-Y, Pharr GM. Effects of drying on the mechanical properties of bovine femur measured by nanoindentation. J Mater Sci Mater Med. 1999;10(8):485-488.
2005	Boskey AL, DiCarlo E, Paschalis E, West P, Mendelsohn R. Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation. Osteoporos Int. December 2005;16(12):2031-2038.
2000	Hoffler CE, Moore KE, Kozloff K, Zysset PK, Goldstein SA. Age, gender, and bone lamellae elastic moduli. J Orthop Res. May 2000;18(3):432-437.
2002	Benezra Rosen V, Hobbs LW, Spector M. The ultrastructure of anorganic bovine bone and selected synthetic hyroxyapatites used as bone graft substitute materials. Biomaterials. February 2002;23(3):921-928.
1985	Rubin CT, Lanyon LE. Regulation of bone mass by mechanical strain magnitude. Calcif Tiss Int. 1985;37(4):411-417.
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2004	Akkus O, Adar F, Schaffler MB. Age-related changes in physicochemical properties of mineral crystals are related to impaired mechanical function of cortical bone. Bone. March 2004;34(4):443-453.
2001	Yamashita J, Furman BR, Rawls HR, Wang X, Agrawal CM. The use of dynamic mechanical analysis to assess the viscoelastic properties of human cortical bone. J Biomed Mater Res. 2001;B58(1):47-53.
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1973	Katz EP, Li S-T. Structure and function of bone collagen fibrils. J Mol Biol. October 15, 1973;80(1):1-15.
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Year

Entry

1994

Mente PL, Lewis JL. Elastic modulus of calcified cartilage is an order of magnitude less than that of subchondral bone. J Orthop Res. September 1994;12(5):637-647.

1984

Lees S, Bonar LC, Mook HA. A study of dense mineralized tissue by neutron diffraction. Int J Biol Macromol. 1984;6(6):321-326.

2000

Boivin GY, Chavassieux PM, Santora AC, Yates J, Meunier PJ. Alendronate increases bone strength by increasing the mean degree of mineralization of bone tissue in osteoporotic women. Bone. November 2000;27(5):687-694.

2007

Lievers WB, Lee V, Arsenault SM, Waldman SD, Pilkey AK. Specimen size effect in the volumetric shrinkage of cancellous bone measured at two levels of dehydration. J Biomech. 2007;40(9):1903-1909.

1996

Landis WJ, Hodgens KJ, Arena J, Song MJ, McEwen BF. Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography. Microsc Res Tech. 1996;33(2):192-202.

1967

Ascenzi A, Bonucci E. The tensile properties of single osteons. Acta Anat. 1967;158(4):375-386.

1963

Hashin Z, Shtrikman S. A variational approach to the theory of the elastic behaviour of multiphase materials. J Mech Phys Solids. March–April 1963;11(2):127-140.

2002

Fan Z, Swadener JG, Rho JY, Roy ME, Pharr GM. Anisotropic properties of human tibial cortical bone as measured by nanoindentation. J Orthop Res. July 2002;20(4):806-810.

2003

Fan Z, Rho J-Y. Effects of viscoelasticity and time-dependent plasticity on nanoindentation measurements of human cortical bone. J Biomed Mater Res. October 1, 2003;A67(1):208-214.

1988

Ascenzi A. The micromechanics versus the macromechanics of cortical bone: a comprehensive presentation. J Biomech Eng. November 1988;110(4):357-363.

2006

Hofmann T, Heyroth F, Meinhard H, Fränzel W, Raum K. Assessment of composition and anisotropic elastic properties of secondary osteon lamellae. J Biomech. 2006;39(12):2282-2294.

1964

Gong JK, Arnold JS, Cohn SH. Composition of trabecular and cortical bone. Anat Rec. 1964;149(3):325-331.

1951

Evans FG, Lebow M. Regional differences in some of the physical properties of the human femur. J Appl Physiol. 1951;3(9):563-572.

1958

Amprino R. Investigations on some physical properties of bone tissue. Acta Anat. 1958;34(3):161-186.

1993

Skedros JG, Bloebaum RD, Bachus KN, Boyce TM. The meaning of graylevels in backscattered electron images of bone. J Biomed Mater Res. January 1993;7(1):47-56.

2003

Ferguson VL, Bushby AJ, Boyde A. Nanomechanical properties and mineral concentration in articular calcified cartilage and subchondral bone. J Anat. August 2003;203(2):191-202.

2000

Carden A, Morris MD. Application of vibrational spectroscopy to the study of mineralized tissues (review). J Biomed Opt. July 2000;5(3):259-268.

1993

Boyde A, Elliott JC, Jones SJ. Stereology and histogram analysis of backscattered electron images: age changes in bone. Bone. May–June 1993;14(3):205-210.

1996

Pidaparti RMV, Chandran A, Takano Y, Turner CH. Bone mineral lies mainly outside collagen fibrils: predictions of a composite model for osternal bone. J Biomech. July 1996;29(7):909-916.

1992

Oliver WC, Pharr GM. An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J Mater Res. 1992;7(6):1564-1583.

1999

Boyde A, Travers R, Glorieux FH, Jones SJ. The mineralization density of iliac crest bone from children with osteogenesis imperfecta. Calcif Tiss Int. March 1999;64(3):185-190.

2000

Jäger I, Fratzl P. Mineralized collagen fibrils: a mechanical model with a staggered arrangement of mineral particles. Biophys J. October 2000;79(4):1737-1746.

1995

Sasaki N, Enyo A. Viscoelastic properties of bone as a function of water content. J Biomech. 1995;28(7):809-815.

2006

Barthelat F, Li C-M, Comi C, Espinosa HD. Mechanical properties of nacre constituents and their impact on mechanical performance. J Mater Sci. 2006;21(8):1977-1986.

2002

Hengsberger S, Kulik A, Zysset P. Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. Bone. 2002;30(1):178-184.

2007

Ramasamy JG, Akkus O. Local variations in the micromechanical properties of mouse femur: the involvement of collagen fiber orientation and mineralization. J Biomech. 2007;40(4):910-918.

2000

Hoffler CE, Moore KE, Kozloff K, Zysset PK, Brown MB, Goldstein SA. Heterogeneity of bone lamellar-level elastic moduli. Bone. June 2000;26(6):603-609.

2004

Oliver WC, Pharr GM. Measurement of hardness and elastic modulus by instrumented indentation: advances in understanding and refinements to methodology. J Mater Res. January 2004;19(1):3-20.

1994

Finlay JB, Hardie WR. Anisotropic contraction of cortical bone caused by dehydration of samples of the bovine femur in vitro. Proc Inst Mech Eng Part H-J Eng Med. 1994;208(1):27-32.

2003

Burr DB, Miller L, Grynpas M, Li J, Boyde A, Mashiba T, Hirano T, Johnston CC. Tissue mineralization is increased following 1-year treatment with high doses of bisphosphonates in dogs. Bone. December 2003;33(6):960-969.

2006

Bembey AK, Bushby AJ, Boyde A, Ferguson VL, Oyen ML. Hydration effects on the micro-mechanical properties of bone. J Mater Res. August 2006;21(8):1962-1968.

1998

Zysset PK, Guo XE, Hoffler CE, Moore KE, Goldstein SA. Mechanical properties of human trabecular bone lamellae quantified by nanoindentation. Technol Health Care. December 1998;6(5-6):429-432.

1999

Zysset PK, Guo XE, Hoffler CE, Moore KE, Goldstein SA. Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur. J Biomech. October 1999;32(10):1005-1012.

1997

Bloebaum RD, Skedros JG, Vajda EG, Bachus KN, Constantz BR. Determining mineral content variations in bone using backscattered electron imaging. Bone. May 1997;20(5):485-490.

2010

Zhang J, Michalenko MM, Kuhl E, Ovaert TC. Characterization of indentation response and stiffness reduction of bone using a continuum damage model. J Mech Behav Biomed Mater. February 2010;3(2):189-202.

2001

Rho JY, Currey JD, Zioupos P, Pharr GM. The anisotropic Young's modulus of equine secondary osteones and interstitial bone determined by nanoindentation. J Exp Biol. May 2001;204(10):1775-1781.

2002

Rho JY, Zioupos P, Currey JD, Pharr GM. Microstructural elasticity and regional heterogeneity in human femoral bone of various ages examined by nano-indentation. J Biomech. February 2002;35(2):189-198.

1991

Lee DD, Glimcher MJ. Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone. J Mol Biol. 1991;217(3):487-501.

2006

Kazanci M, Roschger P, Paschalis EP, Klaushofer K, Fratzl P. Bone osteonal tissues by Raman spectral mapping: orientation–composition. J Struct Biol. December 2006;156(3):489-496.

1964

Currey JD. Three analogies to explain the mechanical properties of bone. Biorheology. 1964;2(1):1-10.

1999

Currey JD. The design of mineralised hard tissues for their mechanical functions. J Exp Biol. December 1999;202(23):3285-3294.

1999

Rho JY, Zioupos P, Currey JD, Pharr GM. Variations in the individual thick lamellar properties within osteons by nanoindentation. Bone. September 1999;25(3):295-300.

1999

Rho J-Y, Pharr GM. Effects of drying on the mechanical properties of bovine femur measured by nanoindentation. J Mater Sci Mater Med. 1999;10(8):485-488.

2005

Boskey AL, DiCarlo E, Paschalis E, West P, Mendelsohn R. Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation. Osteoporos Int. December 2005;16(12):2031-2038.

2000

Hoffler CE, Moore KE, Kozloff K, Zysset PK, Goldstein SA. Age, gender, and bone lamellae elastic moduli. J Orthop Res. May 2000;18(3):432-437.

2002

Benezra Rosen V, Hobbs LW, Spector M. The ultrastructure of anorganic bovine bone and selected synthetic hyroxyapatites used as bone graft substitute materials. Biomaterials. February 2002;23(3):921-928.

1985

Rubin CT, Lanyon LE. Regulation of bone mass by mechanical strain magnitude. Calcif Tiss Int. 1985;37(4):411-417.

1984

Rubin CT, Lanyon LE. Regulation of bone formation by applied dynamic loads. J Bone Joint Surg. March 1984;66A(3):397-402.

2004

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Year	Entry
2013	Paietta RC. Microscale Material Properties of Bone and the Mineralized Tissues of the Intervertebral Disc-Vertebral Body Interface [PhD thesis]. Boulder, CO: University of Colorado; 2013.

Year

Entry

2013

Paietta RC. Microscale Material Properties of Bone and the Mineralized Tissues of the Intervertebral Disc-Vertebral Body Interface [PhD thesis]. Boulder, CO: University of Colorado; 2013.