Mechanical characterisations of Bone

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The ability to determine the mechanical characterizations of bone at microscopic level has a great importance in biological and clinical application. Fatigue and static fracture generally occur due to repeated load or low intensity load for long term rather than trauma, most of the cases of fatigue failure is found in military, athletes, ballet dancer and in young children and frequently increases. The purpose of present study is to examine the mechanical properties of hydrated and non hydrated bovine compact bone using nano-indentation technique. Here we examine elastic modulus, hardness, compressive and tensile strength and their viscoelastic nature by measuring storage and loss modulus of both grouped bones (Hydrated and non hydrated).

Keywords: Nanoindentation, Bone, Hydrated, Bovine, Mechanical property

  1. Introduction

Bone is a composite material whose mechanical performance is strongly dependent on the complex internal hierarchical structure shown in fig 1. At the micron length scale, the mechanical properties of bone are dominated by close interactions between its organic matrix and inorganic mineral component.(Rho, Kuhn-Spearing et al. 1998) Mineral phase (mainly inorganic hydroxypatite) provides the strength and stiffness, and organic matrix (mainly type 1 collagen) provides the softness of bone. While mineral and collagen each contribute to the bone's competency, as do microarchitecture (e.g., porosity and trabecular connectivity), macrostructure (e.g., curvature of diaphysis and thickness of cortical shell), and in vivo microdamage (e.g., microcracks and diffuse cracks) their interaction with water is equally.(Nyman, Roy et al. 2006)

Nanoindentation is widely used in material science. It is a relatively new tool for testing biological tissues. The obtained precise parameters, which have previously been quantified in bone, include hardness and reduced elastic modulus. However In contrast, determination of the viscoelastic properties of bone or mineralized tissues by nanoindentation is less covered.(Pathak, Swadener et al. 2011) Several factors are affecting the mechanical properties of bone: species type, tissue type, age, sample orientation, anatomical location, collagen orientation and degree of mineralization, and these have been studied by nanoindentation as reviewed by. (Feng, Chittenden et al. 2012)

Bone is an inhomogeneous, anisotropic and viscoelastic material, and interaction with water is equally important for the mechanical property of bone. Thus, bone is also a fluidimbibed material in which the distribution of water affects the mechanical properties of bone. The distribution of water in bone appears to change throughout life. It has been reported that water in bone tissues decreases with skeletal growth.(Nyman, Roy et al. 2006) Hence, measurements on the interaction with water at nanoscale add new aspects to the inter-individual biological variation and require precise measurement protocols. To address this issue, the present study investigated the effect of water on the mechanical behavior of goat cortical bone. Specifically nanoindentation technique has been used for studying mechanical properties by using quasistatic and dynamic mechanical analysis.


2. Materials and Methods

2.1. Sample preparation

Tibia bone of goat has been used in this study. The bone has been obtained from local slaughter house and placed in deep freezer at -80oC in solution of ethanol and distilled water. Crossection of bone has been cut into two small pieces using low speed circular saw (Buehler IsoMetTM ). These pieces of samples were divided into two groups. These two sets of samples- hydrated (wet) and dehydrated (dry) were prepared for nanoindentation.

For the wet samples, care was taken to keep the tibia moist with ethanol solution at all times during their preparation and testing. Samples were frozen at -80oC. Dry samples were stored at -5oC after each step of sample preparation and testing. Prior to nanoindentation bone samples of both the group are embedded in epoxy solution (Mixture of EpoThin Resin & EpoThin Hardener) in longitudinal orientation and the mixture was then allowed to cure for 9 hours. . The samples were then grounded and polished using Buehler EcoMet 250 grinder and polisher. Force of 10N was used for this grinding and polishing process. Grinding was done by using sand papers of grit sizes 400, 600 and 1200. For polishing, diamond solutions of 9, 6, 3, 1 and 0.5 µm particle sizes were used.

2.2 Nanoindentation

Nano indentation was performed using a TI-950 TriboIndenter (Hysitron Inc., Minneapolis, MN) equipped with Berkovich tip. Calibration of the instrument was done using standard fused Quartz and polycarbonate sample. A three-sided pyramidal tip with an included angle of 142.3° and a tip radius of ~150 nm was utilized. The testing temperature was 22 °C and relative humidity was 60 %. Regions for testing were identified using an optical microscope integrated into the Nanoindentation system. The “tip to optics calibration” was done by performing 7 indents in “H Pattern”. For area function calibration, a series of indents with different contact depths were performed on standard sample of known elastic modulus, and the contact area was calculated. A plot of the calculated area as a function of contact depth was created and fitted by the TriboScan software.

2.2.1 Quasi Static Indentation

For quasi static analysis of sample, 20 subsequent indents were performed along cross-section of both groups bone samples, specified locations with user-specified parameters as shown in fig 2. Indentations with contact depths of an order of magnitude larger than local surface roughness are thought to be sufficiently deep to avoid a strong effect of roughness on the measured properties. Hence peak load 5000 µN was selected. A load function consisting of a 10s loading to peak force segment, followed by a 30s hold segment and a 10 sec unloading segment were used, shown in figure 3. The hardness and the reduced modulus were automatically computed using the Oliver and Pharr method. The reduced modulusEris related to the young’s modulus Es of testing material through the following relationship:(Herbert, Oliver et al. 2008)




Where, Ei and Ê‹i are the elastic modulus and Poisson’s ratio of indenter material, respectively. Es and Ê‹s are the elastic modulus and Poisson’s ratio for the substrate materials, respectively. Reduced modulus was calculated by taking the value of elastic modulus and Poisson’s ratio to be 1141GPa and 0.07 respectively (for diamond indenter tip).

2.2.2 Dynamic Mechanical Analysis

The dynamic nanoindentation tests were accomplished using the frequency sweep method. Analysis was performed by using nano DMA software in the Histro Triboscope. In this method the maximum applied load is specified to the nanoindentor, probe is swing through a range of frequency (5Hz to 200Hz). By computing the resultant load amplitude, displacement amplitude and phase lag throughout the test, the DMA technique permit the computation of the storage modulus, loss modulus and tan δ using following relation.

Where & are the storage and loss modulus, is the frequency of the applied load, and are the stiffness and damping coefficient of sample and AC is the projected contact area of the indent on the surface of the specimen. (Oyen and Cook 2009)

  1. Result

3.1 Quasi Static Indentation

Comparisons of load displacement plot obtained from quasi static indentation have been shown in fig 4. The elastic moduli were 21.2±4.7 GPa and 12±4.5 GPa for dehydrated and hydrated cortical bone respectively. Variations of elastic moduli from endosteum to periosteum have been shown in fig 5 for dehydrated and hydrated cortical bone. Hardness of dehydrated and hydrated cortical bone was 0.80±0.30 and 0.54±0.36 respectively. Variation of hardness with indent locations has been shown in fig 6 for both groups of bone.

load displacement.jpg

elastic modulus.jpg


Hardness dma.jpg

3.2 Dynamic Mechanical Analysis

DMA is performed on center portion of crosssectional cortical bone. Variation of hardness with frequency was shown in fig 8 for dehydrated and hydrated bone. For comparison of viscoelastic behavior of both group bones, we have done study, storage modulus , loss modulus and their variation with frequency is shown in fig 8 and fig 9 respectively.


loss mod 2c.jpg

4. Conclusion

This study investigated the influence of water on mechanical characterization of goat bone, under quasi static and dynamic mechanical loading. Due to hydration, bone becomes soft as compared to dehydrated bone as shown in fig 4, fig 5 and fig 6(As results obtained from quasi static indentation test). For investigating the effect of hydration from dynamic mechanical analysis, it was found that storage modulus of hydrated bone decrease and loss modulus increase as compared to dehydrated bone, as shown in fig 8 and in fig 9 respectively. From the result of dynamic analysis it was clear that hydrated bone show more viscous behavior.(Pathak, Swadener et al. 2011) Thus water plays an important role for affecting the mechanical property of bone. How it affects the bone at tissue level was not clearly understood. Previous studies suggest that under dry condition no interaction of water with collagen and mineral were present. In the absence of water collagen fibrils might stiffen and contract longitudinally compressing the mineral phase, leading to a higher strength of bone. (Lee, Baldassarri et al. 2012)This statement supports our result.

5. Reference

1. Ebenstein, D. M. and L. A. Pruitt (2006). "Nanoindentation of biological materials." Nano Today 1(3): 26-33.

2. Feng, L., et al. (2012). "Mechanical properties of porcine femoral cortical bone measured by nanoindentation." Journal of biomechanics 45(10): 1775-1782.

3. Herbert, E., et al. (2008). "Nanoindentation and the dynamic characterization of viscoelastic solids." Journal of Physics D: Applied Physics 41(7): 074021.

4. Lee, K.-L., et al. (2012). "Nanomechanical Characterization of Canine Femur Bone for Strain Rate Sensitivity in the Quasistatic Range under Dry versus Wet Conditions." International journal of biomaterials 2012.

5. Menčík, J., et al. (2009). "Determination of viscoelastic–plastic material parameters of biomaterials by instrumented indentation." Journal of the mechanical behavior of biomedical materials 2(4): 318-325.

6. Nyman, J. S., et al. (2006). "The influence of water removal on the strength and toughness of cortical bone." Journal of biomechanics 39(5): 931-938.

7. Oyen, M. L. and R. F. Cook (2009). "A practical guide for analysis of nanoindentation data." Journal of the mechanical behavior of biomedical materials 2(4): 396-407.

8. Pathak, S., et al. (2011). "Measuring the dynamic mechanical response of hydrated mouse bone by nanoindentation." Journal of the mechanical behavior of biomedical materials 4(1): 34-43.

9. Rho, J.-Y., et al. (1998). "Mechanical properties and the hierarchical structure of bone." Medical engineering & physics 20(2): 92-102.