Bone regeneration is a huge challenge in the field of Orthopedic Medicine. Present techniques for the treatment of massive bone loss are fundamentally dependent on artificial prostheses. Prostheses cannot be used for every case due to the limitation of movement and biocompatibility issues. Similarly, after long term use, the prosthesis can fail and result in the loss of function and possibly morbidity.
New nanoscale analysis performed on bones and other mineralized biological materials open a new window into the intricate details of mechanical behavior at very small scales. Micro and macroscopic analysis, which yield averaged quantities over larger length scales, may not be sensitive enough to identify the underlying differences between two similar samples. Hence, nanoscale studies are desirable for the resolved characterization of these complex materials. Furthermore, nanoscale methodologies are beneficial when the volume of material available is too small for larger scale analyses, for instance with tissue engineered bone formation in rat models and critical-sized defects. The accuracy of biomechanical properties reduced using traditional engineering beam theory applied to whole bone bending analysis on mouse bone has also been questioned.
Nanoindentation analysis focuses on differences between trabecular and cortical bone, time dependent plasticity, anisotropy, variations as a function of distance from the osteonal center through the femoral cortex, viscoelasticity and varations due to mineral content.
So as to assess the role of intrinsic bone tissue quality in bone strength, a nanoindentation test was conducted at the level of the vertebral cortex of adult rats after a number of hormonal and dietary manipulations known to significantly impact bone strength of an intact skeletal piece.
The nanoindentation method evaluates both elasticity and hardness of wet and dry bone tissue with a high spatial resolution. Nanoindentation has also been proved to be a reliable technique to evaluate the intrinsic mechanical properties of single bone structural units (BSU). The local elastic properties of bone structural units were found to differ greatly among individuals, the type of bone (osteonal, interstitial, and trabecular), anatomical locations and trabecular orientation.
The results of the current research reveal that besides microarchitecture and geometry, intrinsic bone tissue property is a key determinant of the mechanical competence of rat vertebrae after low protein intake and dietary OVX treatment.
Figure 1. Force-displacement curve of a nanoindentation test: loading (1), holding (2), unloading (3) of an indenter tip. The third part leads to elastic recovery of the material and its initial slope is used to derive the elastic indentation modulus. The hysteresis represents the dissipated energy.
Nanoindentation signifies a test of the fundamental mechanical properties of bone tissue. This method obtains force displacement data of a pyramidal diamond indenter that is pressed into a material. The resulting curve comprising of three parts is shown in Figure 1. In part 1, the indenter tip is loaded onto the sample that leads to a complex combination of elastic and post yield deformation.
At maximum force, the load is held constant leading to creep of the material below the tip. When the force on the tip is released, the elastic reaction of the material is detected (Part 3). The slope at the point of preliminary unloading is considered to derive the elastic properties of the sample. The unloading slope has with:
a direct relationship with the contact area Ac (hmax) and the reduced modulus Er. The contact area is the projected area of contact between the pyramidal indenter and the sample and represents a calibration parameter.
The reduced modulus denotes a sum of the compliance of the material and the diamond indenter.
The first fraction is defined as the indentation modulus and arises from the established properties of the reduced modulus and the indenter tip. The indentation modulus joins with - nucleation density increases with,
the local elastic modulus and Poisson ratio of the specimen and represents the first parameter of interest in this paper. The ratio of maximum force and the contact area supplies a second mechanical parameter, hardness:
Hardness can be understood as a mean pressure the material can resist.
A third output of the indentation experiment was taken into account, i.e., the area of the hysteresis as shown in Figure 1. This parameter has the dimension of mechanical work and signifies the energy dissipated during the indentation test.
Figure 2. Schematic representation of the elasto-plastic behavior on bone during the nanoindentation.
To conduct the nanoindentation tests, the L5 vertebral body of each rat was dissected at the level of the intervertebral disks. The bone specimens were kept frozen until preparation for the mechanical tests. The vertebra was cut transversally in the middle of the 8 mm high body. The samples were embedded in PMMA and the face of the transverse cut was polished finishing with a 0.25 µm diamond solution. After these preparation steps, the specimens were dried for 24 hours at 50 °C.
The mechanical tests included nine indents on the cortical shell of each vertebral body, three indents at the posterior, three at the lateral and three more on the anterior site. On each site, three indents were done on the central, the periosteal and the endosteal location of the bone matrix as shown in Figure 3a. The indents were at 900 nm maximum depth applying an estimated strain rate of e = 0.066 1/seconds for both loading and unloading. At highest load, a 5 second holding period was used. The limit of the maximum allowable thermal drift was set to 0.1 nm/seconds. The indents were performed at the center of the lamellae; indents at the edge of two lamellae were omitted. In the current study, only cortical bone was examined, since huge deterioration and destruction of the trabecular structure was noticed in OVX rats fed a low protein diet.
Figure 3a. Schematic representation of the indent areas. On transversal slices of lumbar vertebral body, three sites were chosen: anterior, posterior, and lateral sites (see left figure). On each site, three locations were defined as structure of interest: the periosteal, central, and endosteal locations (see right part of the figure).
Figure 3b. Optical micrography of Berkovich indentation on Trabecular/ cortical Nanoindentation and AFM high-resolution image. (12 x 12 x 1.5 µm )
Determinants of Bone Strength
Amount of Material
Variation of nanomechanical features in vertebral body cortices
The heterogeneity of nanomechanical features measured in different sites of the vertebral body cortex, (that is, posterior, anterior, and lateral) was first assessed in controls animals. For all three mechanical parameters (hardness, indentations modulus and the dissipated energy) lower values were detected on the anterior site. A two-way ANOVA completed with location and site as fixed effects, revealed that site was very important for all these three mechanical parameters (P < 0.0001). On the other hand, location (endosteal, periosteal, or central) was not important (P > 0.6).
Effect of Protein Intake on Nanomechanical Characteristics
The influence of isocaloric protein undernutrition and of vital amino acids supplements was then evaluated. Three-way ANOVA for indentation modulus considering the complete data set revealed again high universal significance for the site (lateral, anterior, posterior) (P < 0.001). The factor location (endosteal, periosteal, central) was moderately significant (P = 0.029) and treatment was not universally significant (P = 0.65). However, the interaction between treatment and site was near the significance level (P = 0.06). This made the Researchers apply an individual statistical assessment for each of the three sites. Two-way ANOVAs were performed with location and treatment as fixed effects. The impact of the treatment was not substantial (P > 0.1). In contrast, the factor location was substantial for the anterior site (P = 0.013) and for the posterior site (P = 0.0002) but not substantial for the lateral site (P = 0.2). The nano-mechanical properties of the different locations are individually illustrated in Figure 4. Comparison between the treatment groups was performed for all locations as illustrated in Table 1. Post hoc analysis revealed substantial decreases of nanomechanical properties (P < 0.05) at the endosteal location in OVX rats fed the low protein diet as compared with SHAM. This difference was detectable for all three nanomechanical parameters. On the central part of the posterior vertex, dissipated energy and hardness were greatly decreased (P = 0.02 and P = 0.03, respectively) in reaction to ovariectomy and the low protein diet. In periosteal location, significant alteration of energy dissipation and elastic properties between OVX and SHAM rats with the low protein diet was also detected (P = 0.01 and P = 0.02, respectively). The positive trend of essential amino acids supplements on indentation modulus and dissipated energy was not substantial (P < 0.1) at the endosteal location. There was also a trend for an effect of essential amino acid supplements on hardness at the central location (Pb < 0.1). For indentation modulus at the periosteal location, the effects of essential amino acid supplements were nearly significant (P = 0.06).
Macroscopic Mechanical Results Versus Nanomechanical Tissue Properties and Bone Mineral Mass
For the correlation between macroscopic tests and nanomechanical data, which were acquired by axial compression of the vertebral body , the mean values of hardness, indentation modulus and dissipated energy of each rat were used, as shown in Figure 5. Macroscopic energy to failure revealed a correlation (R2 = 0.6) with the dissipated energy of the indentation test. Macroscopic ultimate strength correlated moderately with hardness (R2 = 0.27) and stiffness showed no correlation with the intrinsic elastic properties.
Figure 5. Results of the nanoindentation tests at the level of the posterior part of the vertebral body for all three treatment groups (see text). *Statistically significant ( P < 0.05).
Comparison between axial compression (old macroscopic method) and Nano-indentation (New nanometric method) are perfectly reliable but the nanoindentation give more results on tissue behavior at low scale.
Figure 6. Comparison between axial compression testing and nanoindentation t groups (see text).
The current research revealed a heterogeneity of intrinsic one tissue properties of the rat vertebral body, which differed in relation to protein intake. Low protein intake linked with ovariectomy, paired with vital amino acid supplements, reduced the nanomechanical values. These results highlight the capacity of the nanoindentation method to detect modifications induced by nutritional and hormonal manipulations. Correlations between macroscopic mechanical results as evaluated by axial compression of the vertebral body and nanomechanical tissue properties indicate that macroscopic postelastic behavior differed greatly with material fragility detected on the tissue level. However, macroscopic stiffness was subject by bone geometry modifications and less by variations of tissue properties, as nanoindentation exposes. Other mineralized biological materials such as enamel, dentine and calcified cartilage could be examined by the nanoindentation method.
We thank Dr Patrick Ammann from Service of Bone Diseases [WHO Collaborating Center for Osteoporosis Prevention], Department of Rehabilitation and Geriatrics, 7 University Hospital, Geneva, Switzerland for the use of his complete studies.
Paper : Bone ISSN 8756-3282 2005, vol. 36, no1, pp. 134-141 [8 page(s) (article)] (28 ref.)
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