journal of the mechanical behavior of biomedical materials 46 (2015) 261–270
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Research Paper
Microstructure and compressive mechanical properties of cortical bone in children with osteogenesis imperfecta treated with bisphosphonates compared with healthy children Laurianne Imberta, Jean-Charles Aure´ganb,c, Ke´lig Pernellea, Thierry Hoca,n a
LTDS UMR CNRS 5513, Ecole Centrale Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France Department of Pediatric Orthopedics, Necker—Enfants Malades Hospital, AP-HP, Paris Descartes University, 145 rue de Sèvres, 75014 Paris, France c B2OA UMR CNRS 7052, University Paris-Diderot, 10 avenue de Verdun, 75010 Paris, France b
art i cle i nfo
ab st rac t
Article history:
Osteogenesis imperfecta (OI) is a genetic disorder characterized by a change in bone tissue
Received 26 September 2014
quality, but little data are available to describe the factors involved at the macroscopic
Received in revised form
scale. To better understand the effect of microstructure alterations on the mechanical
12 December 2014
properties at the sample scale, we studied the structural and mechanical properties of six
Accepted 18 December 2014
cortical bone samples from children with OI treated with bisphosphonates and compared
Available online 16 February 2015
them to the properties of three controls. Scanning electron microscopy, high resolution
Keywords:
computed tomography and compression testing were used to assess these properties. More
Osteogenesis imperfecta
resorption cavities and a higher osteocyte lacunar density were observed in OI bone
Mechanical properties
compared with controls. Moreover, a higher porosity was measured for OI bones along with
Porosity
lower macroscopic Young’s modulus, yield stress and ultimate stress. The microstructure
Bone mineral density
was impaired in OI bones; the higher porosity and osteocyte lacunar density negatively
High resolution X-ray computed
impacted the mechanical properties and made the bone more prone to fracture.
tomography
& 2015 Elsevier Ltd. All rights reserved.
Osteocyte lacunae density
1.
Introduction
of disease severity (Sillence et al., 1979), but typical features can be observed in every type. OI includes progressive skeletal
Osteogenesis imperfecta (OI) is a collagen disease caused, in most cases, by a genetic disorder that primarily affects the genes COL1A1 and COL1A2, which encode collagen type I chains. Various forms of OI are associated with different levels n
Corresponding author. Tel.: þ33 472186214. E-mail address: [email protected] (T. Hoc).
http://dx.doi.org/10.1016/j.jmbbm.2014.12.020 1751-6161/& 2015 Elsevier Ltd. All rights reserved.
deformities (Pazzaglia et al., 2013), fractures at all life stages, short stature (Glorieux, 2008; Forlino and Marini, 2000) and often respiratory problems, dentinogenesis imperfecta (Glorieux, 2008) and musculoskeletal manifestations (McKiernan, 2005).
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journal of the mechanical behavior of biomedical materials 46 (2015) 261 –270
More details about the forms of OI can be found in previous publications (Glorieux, 2008; Cundy, 2012). The diagnosis is mainly based on the patient history and a physical examination that considers many factors, such as the family history, the number of fractures or the degree of deformity (Cundy, 2012). A molecular analysis of type I collagen and its encoding genes, mutations screening and bone mineral density (BMD) measurements can help to make the diagnosis (Glorieux, 2008; Rauch and Glorieux, 2004). In particular, the BMD measured by DXA was lower in OI bones (Cepollaro et al., 1999; Reinus et al., 1998). Although studies have examined treatments that utilize grafting and mesenchymal stem cells (Liu et al., 2014), curative treatments do not exist and bisphosphonates act only to strengthen the bones by reducing the resorbing activity (Ebetino et al., 2011). These medicines, which are administered orally (Ward et al., 2010; Vyskočil et al., 2005) or intravenously (Williams et al., 1997; Shapiro et al., 2003), are commonly prescribed for children suffering from OI. This treatment significantly decreases the fracture rate and improves ambulation or mobility (Ward et al., 2010; Vyskočil et al., 2005). Bone tissue quality is well known to be impaired in both the OI mice (oim) model (Vanleene et al., 2012, 2011; Coleman et al., 2012; Yao et al., 2013; Camacho et al., 2003; Fratzl et al., 1996; Grabner et al., 2001) and in human tissues (Pazzaglia et al., 2013; Cepollaro et al., 1999; Reinus et al., 1998; Rauch et al., 2005, 2000; Jones et al., 1999; Roschger et al., 2008; Boyde et al., 1999; Sarathchandra et al., 1999, 1999, 2000; Vetter et al., 1991; Fratzl-Zelman et al., 2014). In particular, the morphology of collagen fibers was altered; the fibers were smaller in diameter and disorganized (Sarathchandra et al., 1999, 2000). These changes led to mineralization abnormalities, such as patchy mineralization (Sarathchandra et al., 2000) and smaller hydroxyapatite crystals (Fratzl et al., 1996; Vetter et al., 1991; Fratzl-Zelman et al., 2014; Imbert et al., 2014). As a consequence, the mechanical properties at the microscopic scale measured by nanoindentation in mice (Vanleene et al., 2012, 2011) or in children (Imbert et al., 2014) were lower in bone affected by OI, and they negatively correlated with disease severity (Albert et al., 2013; Fan et al., 2007). At the macroscopic scale, a few studies have investigated the effects of OI on the microstructure of human bones. In particular, they showed abnormal remodeling activity (Pazzaglia et al., 2013; Cepollaro et al., 1999; Rauch et al., 2000; Roschger et al., 2008; Braga et al., 2004), which led to a lower osteon density (Pazzaglia et al., 2013; Jones et al., 1999) and the formation of “atypical” cavities (Pazzaglia et al., 2013). However, the effects of microstructure alterations on the macroscopic mechanical properties have only been partially investigated. Recently, Albert et al. (2014) linked the intracortical vascular porosity measured by synchrotron tomography to mechanical properties measured by three-point bending in the longitudinal direction, but they did not find a significant correlation in the transverse direction. To date and to our knowledge, a direct experimental comparison of mechanical properties at the sample scale from OI and healthy children has not been performed, and the link with the mechanical properties was only partially investigated if a microstructural abnormality was reported in the literature. Thus, the aim of this study was twofold: (i) compare the mechanical and
microstructural properties between OI treated with bisphosphonates and healthy children and (ii) establish correlations between mechanical and microstructural properties within the OI population to determine the major factors involved in the OI bone fragility. The osteocyte lacunar density, vascular porosity and macroscopic mechanical properties were assessed with scanning electron microscopy, high resolution X-ray computed tomography and compression testing, respectively.
2.
Materials and methods
2.1.
Samples
Bone specimens were collected over one year from young individuals with and without OI pathology. The OI donors were five pediatric patients (two males, three females) between 7 and 21 years old with mild to severe OI who underwent routine surgery at Necker Hospitals for Children— Paris for fracture repair or deformity correction of the lower extremity long bones. They sustained a minimum of seven fractures and were all treated with pamidronate (Aredias) therapy. The control group consisted of three donors (one male, two females) without bone pathology who underwent routine surgery at the Necker Hospitals for Children—Paris for fracture repair. The donor ages and BMI did not differ significantly between the two groups (p¼ 0.55 and p¼ 0.85, respectively). Clinical data about the patients are provided in Table 1. Six cortical bone specimens from OI donors and three cortical bone specimens from healthy donors were collected from long bones. Two samples originated from the same patient, but they were from both femurs in a patient who underwent surgery twice, two months apart. The study was conducted according to the Institutional Review Board recommendations (IRB 00003835 2010/28NI). The samples were stored at 20 1C until testing. All bone samples were cut with a diamond saw (Secotom-15, Struers A/S, Ballerup, Denmark) into small parallelepipeds. The size of the specimen depended on the amount of bone and was approximately 3 mm long, 1.6 mm wide and 1.6 mm high. The samples were prepared for transversal testing, i.e., in the direction perpendicular to the osteonal axis.
Table 1 – Clinical data of the control patients and OI patients type III treated with pamidronate. Samples
Sex
Age
Weight
OI 1 OI 2 OI 3 OI 4 OI 5 OI 6 Control 1 Control 2 Control 3
M F M M F F M F F
13 11 14 14 7 21 14 8 16
39 24 53 53 26 30 50 22 46
journal of the mechanical behavior of biomedical materials 46 (2015) 261 –270
2.2.
High-resolution X-ray computed tomography
Each bone sample was immersed in a physiological saline solution (NaCl 0.9%) within a cryotube and imaged using high resolution X-ray computed tomography (Phoenix Nanotom S, GE Sensing & Inspection Technologies GmbH, Wunstorf, Germany) equipped with a high-power nanofocus tube with a molybdenum target. Projection images on a CCD camera were obtained at 70 kV and 130 μA with a resolution of 4 mm. A rotation of 0.181 was used between each image acquisition, providing a series composed of 2000 projection images. The software Phoenix Datosx 2 (GE Sensing & Inspection Technologies GmbH, Wunstorf, Germany) was used to reconstruct a stack of 2-D sections from this series of projection images for each bone sample. Beam hardening correction was applied during reconstruction using a method developed by GE Healthcare (GE Sensing and Inspection Technologies GmbH, 2010). The stacks were stored as TIFF files, with indexed grey levels ranging from 0 (black) to 255 (white). Two phantoms with 0.25 g/cm3 and 0.75 g/cm3 of hydroxyapatite for bone density calibration were imaged at the same time as the bone samples. Their mean grey level was defined as the maximum of the grey level distribution. The CT-Analyzer software (Bruker-microCT, Kartuizersweg 3B, Belgium) was used to calculate the vascular porosity (including Haversian canals, Volkmann’s canals and all open and closed cavities with a diameter larger than 25 mm) and the bone mineral density (BMD in gHA/cm3) of the entire sample, including the porosity. To discriminate between the porosity and the tissue, the threshold was chosen as the grey level corresponding to the minimum between the two modes of the bimodal distribution (Chappard et al., 2008). The tissue mineral density (TMD in gHA/cm3) was calculated as the BMD divided by (1—porosity).
2.3.
263
surface perpendicular to the osteonal axis was imaged for each sample with a Quanta 250 FEG microscope (FEI, Hillsboro, Oregon, USA) equipped with a GDA detector in environmental conditions. Thus, sample preparation was not required. The microscope was operated at a voltage of 30 kV and hydrostatic pressure of 0.9 Torr (120 Pa) in the chamber. To reflect the difference in mineralization (Roschger et al., 1998, 2008; Fratzl-Zelman et al., 2009), acquisitions were performed in the backscattered electron mode. A magnification of 50 and a very slow scanning (30 ms per point) were used to obtain an image of the maximum surface with an optimal contrast. The resulting images were in 24 bit TIF format with a resolution of 4096 3536 pixel. For each sample, the number of osteocyte lacunae was counted manually from the SEM images with a superimposed grid (Fig. 1). Based on these images, thresholding was performed manually with the free software imageJ to separate the tissue (in white) from the porosity in order to have the macroscopic porosity in black (Fig. 1). The tissue surface was defined as the image area minus the porosity area, and the osteocyte lacunar density (#/mm2) was calculated as the ratio of the lacunae number over the tissue area.
Compression testing
Each hydrated sample was tested in a homemade compression machine that was controlled by a LabView acquisition card (National Instrument Corporation, Austin, Texas, US). The sample was placed between two cylindrical jaws and maintained by a 10 N pre-load. A displacement rate of 0.7 mm/ s was applied to the mobile jaw. The test consisted of three steps; first, the sample was loaded until 100 N, and then the sample was unloaded and loaded again to fracture. The load and displacement were registered during the test by means of an extensometer and a 2.5 kN load cell. The stress-strain curve was derived from the load-displacement curve by normalizing the load by the initial section and the displacement by the initial length. The macroscopic Young’s modulus (E) was defined as the slope of the linear unloading portion. The inflection point at the boundary between the linear and non-linear domains was defined as the yield stress (σy). The ultimate stress (σmax) was defined as the maximum stress reached during the test, and the ultimate strain was the strain associated with this stress.
2.4. Scanning electron microscopy and osteocyte lacunae counting Scanning electron microscopy (SEM) was used to image the sample after compression testing. In the present study, a
Fig. 1 – (a) SEM image of a OI sample (OI 4) with a superimposed grid (b) binary image made via thresholding from (a) without grid.
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2.5.
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Statistical analysis
A statistical analysis was performed using R (The R foundation) with a threshold of 5% corresponding to the alpha risk. A nonparametric Mann–Whitney test was used to detect any significant differences between the parameters of the pathological bones and the controls. The influence of the porosity on the mechanical properties was studied using a Spearman correlation.
3.
Results
3.1.
Microstructure observations
Two scales of porosity were observed on these figures: (i) the vascular intracortical porosity, which corresponds to the Haversian canals in healthy bones and present larger cavities in OI samples, and (ii) the osteocyte lacunar porosity, with a higher number of lacunae in OI bone. To quantify this effect, the osteocyte lacunar density (number of osteocyte lacunae per area) was measured. The density was significantly higher for OI samples (6407195.5 #/mm2) compared with controls (395.8715.8 #/mm2, p¼ 0.024) (Fig. 5). Based on the SEM images of samples after the compression tests, the crack paths also differed between OI and control bones. Based on observations, a major crack appeared on all control samples, whereas smaller but more numerous cracks were observed on the OI samples surface.
Scanning electron microscopy in backscattered electron mode was used to image the samples after compression testing. Figs. 2–4 show the microstructures of cortical bones for healthy and diseased children.
Fig. 2 – SEM images of the three controls (C1, C2, C3 from top to bottom) taken after compression test.
Fig. 3 – SEM images of three OI samples (OI 1, OI 2, OI 3 from top to bottom) taken after compression test.
journal of the mechanical behavior of biomedical materials 46 (2015) 261 –270
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Fig. 4 – SEM images of two OI samples (OI 5, OI 6 from top to bottom) taken after compression test.
Fig. 6 – 3D images of (a) a control and (b) a OI reconstructed from the tomography acquisitions. The left part of each sample is inverted to show only the porosity.
Fig. 5 – Comparison of osteocyte lacunar density between the control group and the OI group (npo0.05).
3.2.
Vascular intracortical porosity
The vascular intracortical porosity was measured using high resolution X-ray computed tomography. The difference in the vascular intracortical porosity was observed on 3D images reconstructed from the tomography acquisitions, for which one part of the sample was inverted to reveal the porosity. Fig. 6 shows the typical 3D images of control and OI samples reconstructed from the tomography acquisitions. The cavities visible on the surface
on SEM images were present in the entire structure. The porosity microarchitecture, which consisted of thin channels in control bone, was replaced by a rougher architecture in OI bone. The values of the vascular intracortical porosity for each sample are given in Table 2. As expected, the porosity was significantly higher in OI samples compared with controls.
3.3. Bone mineral density (BMD) and tissue mineral density (TMD) The bone mineral density (the density in mineral (hydroxyapatite HA) of the entire sample including the porosity) was measured based on the tomography acquisitions. The results are given in Table 2. The BMD of OI bones was significantly lower than that of control bones. On the contrary, the TMD was significantly higher in OI samples than in controls. The porosity and BMD were significantly negatively correlated (r2 ¼0.89, p¼ 0.017).
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Table 2 – Values of structural parameters (BMD, porosity and TMD) and mechanical parameters (compressive Young’s modulus, ultimate strength, ultimate strain and yield strength) for each sample. Samples
BMD (gHA/cm3)
Porosity (%)
TMD (gHA/cm3)
E (GPa)
σmax (MPa)
σy (MPa)
OI 1 OI 2 OI 3 OI 4 OI 5 OI 6 Mean SD Control 1 Control 2 Control 3 Mean SD
0.621 0.859 0.771 0.909 0.854 0.901 0.819a 0.109 0.918 0.941 0.998 0.952 0.041
45.2 16.6 33 16.5 24.6 16.7 25.4a 11.7 6 2.7 2.2 3.6 2.1
1.133 1.030 1.151 1.089 1.133 1.082 1.103a 0.045 0.977 0.967 1.020 0.988 0.028
3.9 3.3 4 4.7 2.5 5.5 4.0a 1.0 10.5 9.6 6.8 9.0 1.9
49.1 78.6 58.9 87.9 51.7 79 67.5a 16.3 114.2 163.9 151.9 143.3 25.9
33 57 49 70 38 60 51a 14 90 105 80 92 13
BMD¼ bone mineral density, TMD ¼ tissue mineral density. E ¼ Young’s modulus, σmax ¼ultimate stress, and σy ¼yield stress. a Significant difference with the mean values of the control population with a threshold of 5%.
Fig. 7 – Typical stress strain curve for a OI sample.
3.4.
Macroscopic mechanical properties
A typical stress strain curve is presented in Fig. 7, there is a first loading part, then a linear unloading part used to derive the Young’s modulus. Besides, after the maximum stress the stress decreased progressively due to microcracks formation. The values of Young’s modulus, yield stress and ultimate stress, which were derived from the stress–strain curves obtained during the compression tests, are presented in Table 2. These three mechanical parameters were significantly lower in OI samples than in controls. The yield stress and ultimate stress linearly correlated with porosity in OI samples (r2 ¼0.78, p¼ 0.33 for both) (Table 3). Similarly, the yield stress and ultimate stress linearly correlated with BMD (r2 ¼ 0.89, p¼ 0.017 for both) with a slope opposite to the slope of the correlation obtained for the porosity. Furthermore, the Young’s modulus and the microstructural parameters did not correlate, i.e., BMD, TMD, or porosity. Similarly, the
ultimate strain, which corresponds to the strain measured at the ultimate stress point, did not correlate with these microstructural parameters. Notably, none of these correlations were significant for the control group due to the low number of controls; however, the trend between the ultimate stress and porosity was the same with a higher slope.
4.
Discussion and conclusions
In the present study, the mechanical properties and microstructural parameters of bone tissue from patients with OI and controls were analyzed at the macroscopic (sample) level with compression testing, high-resolution X-ray computed tomography and scanning electron microscopy. All samples retrieved from healthy children or patients with OI originated from the long bones, irrespective of the gender distribution. Due to the difficulty in retrieving bone samples from children
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Table 3 – Correlations between the mechanical properties and the microstructure parameters within the OI group. Mechanical properties
Microstructure parameters
Young’s modulus
Vascular intracortical porosity BMD TMD Osteocyte lacunar density
Yield stress
Ultimate stress
R
p-Value
0.02 2.12 3.82 0.0001
0.257 0.486 0.145 0.086
0.65 0.35 0.78 0.91
Vascular intracortical porosity BMD TMD Osteocyte lacunar density
0.9687 99.614 191.53 0.0118
0.886 0.943 0.580 0.029
0.033 0.016 0.22 1
Vascular intracortical porosity BMD TMD Osteocyte lacunar density
1.17 113.7 276.6 0.0026
0.886 0.943 0.580 0.029
0.03 0.016 0.22 1
and the scarcity of the pathology, the number of samples remained low. In the present study, all patients with OI were treated with bisphosphonate (BP) therapy. Although the pathological and healthy groups differed in two ways, our results were clinically relevant because BPs are a common treatment for OI. BPs have been proven to prevent fractures and increase mineral density (Glorieux, 2008; Cundy, 2012; Vyskočil et al., 2005; Shapiro et al., 2003). Boyde et al. (1999), Roschger et al. (2008) showed that the mineral density increased in OI patients without BPs; thus, the trend in the tissue mineral density observed in our study was correct, and the effect of the pathology was simply overestimated. Similarly, BPs decreased the cortical porosity (Shapiro et al., 2003; Roschger et al., 2001), and the trend observed in the present study was correct with underestimated values. To the best of our knowledge, only one very recent study performed macroscopic mechanical tests on bones from children treated with BPs and untreated controls to assess the effects of BPs on the mechanical properties of OI. In this study, Albert et al. (2014) did not draw conclusions about the effects of BPs on the mechanical properties because they only examined two nontreated samples and the strength of one sample fell within the range of treated samples. In a previous study, we investigated the tissue level and we found that the mechanical properties were lower in OI samples treated with bisphosphonates compared to control samples. Besides, the tissue mineral density was higher in OI samples, correlated to the tissue Young’s modulus measured by nanoindentation, unlike the crystallinity (Imbert et al., 2014). Bisphosphonates might have an effect on the mineral nanostructure and the mechanical properties measured by nanoindentation on osteoporotic bones (Bala et al., 2012) but no similar results on OI patients were found. The effects of BPs on OI bones and on osteoporotic bones are likely to be different. The microstructures observed with SEM using the backscattered electron mode differed between healthy and pathological bones. In particular, the osteocyte lacunar density was calculated and found to be significantly higher in OI bones compared with control bones. Many authors have been interested in osteocyte lacunar density in OI children (Albert et al., 2014), healthy animals (Nicolella et al., 2006; Ma et al., 2008) or healthy human adults (Qiu et al., 2005;
Slope
Vashishth et al., 2000; Dong et al., 2010; Carter et al., 2013). The osteocyte lacunae correspond to osteoblasts embedded in the matrix, therefore their number is linked to osteoblast activity. The increased number of osteocyte lacunae is in agreement with an increased number of osteoblast described in the paper of Rauch et al. (2000). Moreover values found in the present study were on the same order of magnitude as results reported in the literature. Others have qualitatively observed a difference in the osteocyte lacunar density in human OI bones (Rauch et al., 2000; Jones et al., 1999) without direct comparison to control bones. To our knowledge, the only quantitative 3D analysis that compared OI and controls was performed for a mouse model (Carriero et al., 2014). As in the present study, the results showed a significant increase in the osteocyte lacunar density for oim compared with wildtype mice (127 365 vs. 72 981 #/mm3) (Carriero et al., 2014). Moreover, larger and more numerous cavities were observed in OI bones. In our study, these large cavities were indicated by the vascular intracortical porosity, which was measured by high-resolution computed tomography and significantly higher in OI samples compared with controls. To date and to our knowledge, the data on cortical porosity in human OI bone are scarce. Albert et al. (2014) measured the vascular intracortical porosity of nine OI donors and found values of 21710%, which agreed with our results. Pazzaglia et al. obtained a higher fraction of the vascular/resorption area in children with OI (between three and eight years) compared with controls (44.3% vs. 13.6%) (Glorieux, 2008). In the oim study, the vascular porosity was measured by tomography (Coleman et al., 2012; Carriero et al., 2014), and Carriero et al. (2014) recently did not find any significant difference in the cortical porosity between oim and wild-type mice (7.8 (2.7) vs. 6.1 (4.6)). A combination of a higher turnover (Cepollaro et al., 1999; Rauch et al., 2000), a slower bone formation due to fewer efficient osteoblasts (Rauch et al., 2000; Roschger et al., 2008) and higher resorption parameters (Rauch et al., 2000; Roschger et al., 2008) may lead to resorption that is not followed by formation, which may explain the formation of these cavities. This modification in the bone remodeling process leading to a modification of osteonal microstructure could not be transposed to the homogeneous tissue of mice. Moreover, the difference in the vascular and lacunar porosities could explain the shorter cracks observed in OI. The cracks did not propagate as well as in controls
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because they met cavities more frequently during propagation. Ma et al. (2008) already reported a negative correlation between the osteocyte density and micro-crack length in rats, suggesting that lacunae are able to stop or initiate cracks in order to dissipate energy. The increased lacunar density might be a way to compensate for the material fragility. Other authors observed a relationship between the osteocyte lacunar density and the micro-crack density (Vashishth et al., 2000) but could not conclude the cause. We hypothesized that the poor mineral quality (Imbert et al., 2014) increased the crack density, and these cracks caused the death of some osteocytes by disrupting the supply of nutrition to these cells, which then increased bone remodeling turnover. Knowledge of the living osteocyte density compared with the osteocyte lacunar density should allow the validation of this assumption. As mentioned by others (Reinus et al., 1998), an increase in the porosity led to a decrease in bone mineral density (BMD). As expected, the BMD measured by high-resolution computed tomography was significantly lower in OI bones compared with controls. At the tissue scale, the tissue mineral density, derived from BMD and porosity, was significantly higher in OI bones compared with controls. This result agreed with previous studies showing that OI bone was more mineralized than normal bone in humans (Roschger et al., 2008; Boyde et al., 1999) and mice (Vanleene et al., 2012; Grabner et al., 2001). The macroscopic Young’s modulus, yield stress and ultimate stress were measured during compression tests and were significantly lower in OI subjects compared with controls. In the literature, the mechanical properties of bones from patients with OI were mainly measured at the tissue scale by nanoindentation (Imbert et al., 2014; Albert et al., 2013; Fan et al., 2007), but few mechanical data at the sample scale exist, and direct comparisons of the mechanical properties at this sample scale between OI and healthy children have not been performed. In the present study, the results obtained for the bones of healthy children were consistent with results obtained in several previous works (Berteau et al., 2014; Öhman et al., 2011). Albert et al. recently performed three-point bending tests on OI cortical bones that required linear elastic beam theory to extract the mechanical parameters. They calculated mean values of 1.670.4 GPa, 20.876 MPa and 26.578.6 MPa for the Young’s modulus, the yield Stress and the ultimate stress, respectively, in the transverse direction (Albert et al., 2014). These results are clearly weaker than the results obtained from compression tests in the present study. The difference could be explained by the nature of the three-point bending test; the stress field is not uniform and mixes the tensile and compression area. The yield and ultimate stresses are strongly sensitive to these two types of loading (Bayraktar et al., 2004). Three-point bending data have also been reported for the oim model (Vanleene et al., 2012, 2011; Yao et al., 2013). However, Vanleene et al. (2011) did not measure a significant difference in the Young’s modulus between oim and wild-type mice (7.071.2 GPa vs. 6.970.7 GPa). Their values were higher than the values measured in humans, proving again that the macrostructure should be taken into account for human samples.
In this study, we aimed to investigate the relationships between the mechanical properties and the microstructure parameters within the OI population in order to identify the factors responsible for OI bone fragility. We found a strong linear correlation between the stresses (yield stress and ultimate stress) and the microstructural parameters, such as the bone mineral density and porosity. The BMD, which takes into account the porosity and tissue mineral, density explained 89% of yield and ultimate stresses, while porosity alone explained 78% of these mechanical parameters. These results showed that the tissue quality was not the only factor responsible for the fragility and that porosity played a significant role. In the literature some studies investigated the microstructure–mechanical properties relationship in bovine (Li et al., 2013) or human (Ks and Nicolella, 2012) bones but, to our knowledge, only one study (Albert et al., 2014) has attempted to identify correlations in OI bones and found no significant correlation between the mechanical properties calculated in the transversal direction and the microstructural parameters. The effect of porosity on the mechanical properties was also investigated for the oim model. For example, Carriero et al. (2014) showed that regions prone to fracture were located mostly around canals. In conclusion, this study measured the structural and mechanical parameters of cortical bone samples from children with OI and compared these values to those measured in controls. The results showed that the mechanical properties were significantly lower in OI bones, while the porosity was significantly higher. The correlation between both was strong but limited, which proved the influence of the tissue quality. The osteocyte lacunar density was significantly higher in OI bones, suggesting that remodeling was altered in OI bones. Although the mechanical properties were found to negatively correlate with the porosity, we did not find a correlation between the osteocyte lacunar density and the mechanical properties. Further investigations from a biological perspective are required to establish a relationship between the osteocyte lacunar density and the bone fragility.
Acknowledgments The authors are grateful to G. Finidori and S. Avril for their helpful discussion. We would also like to thank the engineers and technicians B. Ponsard, O. Pollet, C. Bosser and B. Jeanpierre for their technical support. We also wish to thank the Rhône-Alpes Region, the IVTV ANR-10-EQPX-06-01, ANR OMBIOS and the AOI (Association de l’Ostéogénèse Imparfaite) (72 ANR OMBIOS 11-BS09-036-01) for their financial support.
r e f e r e n c e s
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Research Paper
Microstructure and compressive mechanical properties of cortical bone in children with osteogenesis imperfecta treated with bisphosphonates compared with healthy children Laurianne Imberta, Jean-Charles Aure´ganb,c, Ke´lig Pernellea, Thierry Hoca,n a
LTDS UMR CNRS 5513, Ecole Centrale Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France Department of Pediatric Orthopedics, Necker—Enfants Malades Hospital, AP-HP, Paris Descartes University, 145 rue de Sèvres, 75014 Paris, France c B2OA UMR CNRS 7052, University Paris-Diderot, 10 avenue de Verdun, 75010 Paris, France b
art i cle i nfo
ab st rac t
Article history:
Osteogenesis imperfecta (OI) is a genetic disorder characterized by a change in bone tissue
Received 26 September 2014
quality, but little data are available to describe the factors involved at the macroscopic
Received in revised form
scale. To better understand the effect of microstructure alterations on the mechanical
12 December 2014
properties at the sample scale, we studied the structural and mechanical properties of six
Accepted 18 December 2014
cortical bone samples from children with OI treated with bisphosphonates and compared
Available online 16 February 2015
them to the properties of three controls. Scanning electron microscopy, high resolution
Keywords:
computed tomography and compression testing were used to assess these properties. More
Osteogenesis imperfecta
resorption cavities and a higher osteocyte lacunar density were observed in OI bone
Mechanical properties
compared with controls. Moreover, a higher porosity was measured for OI bones along with
Porosity
lower macroscopic Young’s modulus, yield stress and ultimate stress. The microstructure
Bone mineral density
was impaired in OI bones; the higher porosity and osteocyte lacunar density negatively
High resolution X-ray computed
impacted the mechanical properties and made the bone more prone to fracture.
tomography
& 2015 Elsevier Ltd. All rights reserved.
Osteocyte lacunae density
1.
Introduction
of disease severity (Sillence et al., 1979), but typical features can be observed in every type. OI includes progressive skeletal
Osteogenesis imperfecta (OI) is a collagen disease caused, in most cases, by a genetic disorder that primarily affects the genes COL1A1 and COL1A2, which encode collagen type I chains. Various forms of OI are associated with different levels n
Corresponding author. Tel.: þ33 472186214. E-mail address: [email protected] (T. Hoc).
http://dx.doi.org/10.1016/j.jmbbm.2014.12.020 1751-6161/& 2015 Elsevier Ltd. All rights reserved.
deformities (Pazzaglia et al., 2013), fractures at all life stages, short stature (Glorieux, 2008; Forlino and Marini, 2000) and often respiratory problems, dentinogenesis imperfecta (Glorieux, 2008) and musculoskeletal manifestations (McKiernan, 2005).
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journal of the mechanical behavior of biomedical materials 46 (2015) 261 –270
More details about the forms of OI can be found in previous publications (Glorieux, 2008; Cundy, 2012). The diagnosis is mainly based on the patient history and a physical examination that considers many factors, such as the family history, the number of fractures or the degree of deformity (Cundy, 2012). A molecular analysis of type I collagen and its encoding genes, mutations screening and bone mineral density (BMD) measurements can help to make the diagnosis (Glorieux, 2008; Rauch and Glorieux, 2004). In particular, the BMD measured by DXA was lower in OI bones (Cepollaro et al., 1999; Reinus et al., 1998). Although studies have examined treatments that utilize grafting and mesenchymal stem cells (Liu et al., 2014), curative treatments do not exist and bisphosphonates act only to strengthen the bones by reducing the resorbing activity (Ebetino et al., 2011). These medicines, which are administered orally (Ward et al., 2010; Vyskočil et al., 2005) or intravenously (Williams et al., 1997; Shapiro et al., 2003), are commonly prescribed for children suffering from OI. This treatment significantly decreases the fracture rate and improves ambulation or mobility (Ward et al., 2010; Vyskočil et al., 2005). Bone tissue quality is well known to be impaired in both the OI mice (oim) model (Vanleene et al., 2012, 2011; Coleman et al., 2012; Yao et al., 2013; Camacho et al., 2003; Fratzl et al., 1996; Grabner et al., 2001) and in human tissues (Pazzaglia et al., 2013; Cepollaro et al., 1999; Reinus et al., 1998; Rauch et al., 2005, 2000; Jones et al., 1999; Roschger et al., 2008; Boyde et al., 1999; Sarathchandra et al., 1999, 1999, 2000; Vetter et al., 1991; Fratzl-Zelman et al., 2014). In particular, the morphology of collagen fibers was altered; the fibers were smaller in diameter and disorganized (Sarathchandra et al., 1999, 2000). These changes led to mineralization abnormalities, such as patchy mineralization (Sarathchandra et al., 2000) and smaller hydroxyapatite crystals (Fratzl et al., 1996; Vetter et al., 1991; Fratzl-Zelman et al., 2014; Imbert et al., 2014). As a consequence, the mechanical properties at the microscopic scale measured by nanoindentation in mice (Vanleene et al., 2012, 2011) or in children (Imbert et al., 2014) were lower in bone affected by OI, and they negatively correlated with disease severity (Albert et al., 2013; Fan et al., 2007). At the macroscopic scale, a few studies have investigated the effects of OI on the microstructure of human bones. In particular, they showed abnormal remodeling activity (Pazzaglia et al., 2013; Cepollaro et al., 1999; Rauch et al., 2000; Roschger et al., 2008; Braga et al., 2004), which led to a lower osteon density (Pazzaglia et al., 2013; Jones et al., 1999) and the formation of “atypical” cavities (Pazzaglia et al., 2013). However, the effects of microstructure alterations on the macroscopic mechanical properties have only been partially investigated. Recently, Albert et al. (2014) linked the intracortical vascular porosity measured by synchrotron tomography to mechanical properties measured by three-point bending in the longitudinal direction, but they did not find a significant correlation in the transverse direction. To date and to our knowledge, a direct experimental comparison of mechanical properties at the sample scale from OI and healthy children has not been performed, and the link with the mechanical properties was only partially investigated if a microstructural abnormality was reported in the literature. Thus, the aim of this study was twofold: (i) compare the mechanical and
microstructural properties between OI treated with bisphosphonates and healthy children and (ii) establish correlations between mechanical and microstructural properties within the OI population to determine the major factors involved in the OI bone fragility. The osteocyte lacunar density, vascular porosity and macroscopic mechanical properties were assessed with scanning electron microscopy, high resolution X-ray computed tomography and compression testing, respectively.
2.
Materials and methods
2.1.
Samples
Bone specimens were collected over one year from young individuals with and without OI pathology. The OI donors were five pediatric patients (two males, three females) between 7 and 21 years old with mild to severe OI who underwent routine surgery at Necker Hospitals for Children— Paris for fracture repair or deformity correction of the lower extremity long bones. They sustained a minimum of seven fractures and were all treated with pamidronate (Aredias) therapy. The control group consisted of three donors (one male, two females) without bone pathology who underwent routine surgery at the Necker Hospitals for Children—Paris for fracture repair. The donor ages and BMI did not differ significantly between the two groups (p¼ 0.55 and p¼ 0.85, respectively). Clinical data about the patients are provided in Table 1. Six cortical bone specimens from OI donors and three cortical bone specimens from healthy donors were collected from long bones. Two samples originated from the same patient, but they were from both femurs in a patient who underwent surgery twice, two months apart. The study was conducted according to the Institutional Review Board recommendations (IRB 00003835 2010/28NI). The samples were stored at 20 1C until testing. All bone samples were cut with a diamond saw (Secotom-15, Struers A/S, Ballerup, Denmark) into small parallelepipeds. The size of the specimen depended on the amount of bone and was approximately 3 mm long, 1.6 mm wide and 1.6 mm high. The samples were prepared for transversal testing, i.e., in the direction perpendicular to the osteonal axis.
Table 1 – Clinical data of the control patients and OI patients type III treated with pamidronate. Samples
Sex
Age
Weight
OI 1 OI 2 OI 3 OI 4 OI 5 OI 6 Control 1 Control 2 Control 3
M F M M F F M F F
13 11 14 14 7 21 14 8 16
39 24 53 53 26 30 50 22 46
journal of the mechanical behavior of biomedical materials 46 (2015) 261 –270
2.2.
High-resolution X-ray computed tomography
Each bone sample was immersed in a physiological saline solution (NaCl 0.9%) within a cryotube and imaged using high resolution X-ray computed tomography (Phoenix Nanotom S, GE Sensing & Inspection Technologies GmbH, Wunstorf, Germany) equipped with a high-power nanofocus tube with a molybdenum target. Projection images on a CCD camera were obtained at 70 kV and 130 μA with a resolution of 4 mm. A rotation of 0.181 was used between each image acquisition, providing a series composed of 2000 projection images. The software Phoenix Datosx 2 (GE Sensing & Inspection Technologies GmbH, Wunstorf, Germany) was used to reconstruct a stack of 2-D sections from this series of projection images for each bone sample. Beam hardening correction was applied during reconstruction using a method developed by GE Healthcare (GE Sensing and Inspection Technologies GmbH, 2010). The stacks were stored as TIFF files, with indexed grey levels ranging from 0 (black) to 255 (white). Two phantoms with 0.25 g/cm3 and 0.75 g/cm3 of hydroxyapatite for bone density calibration were imaged at the same time as the bone samples. Their mean grey level was defined as the maximum of the grey level distribution. The CT-Analyzer software (Bruker-microCT, Kartuizersweg 3B, Belgium) was used to calculate the vascular porosity (including Haversian canals, Volkmann’s canals and all open and closed cavities with a diameter larger than 25 mm) and the bone mineral density (BMD in gHA/cm3) of the entire sample, including the porosity. To discriminate between the porosity and the tissue, the threshold was chosen as the grey level corresponding to the minimum between the two modes of the bimodal distribution (Chappard et al., 2008). The tissue mineral density (TMD in gHA/cm3) was calculated as the BMD divided by (1—porosity).
2.3.
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surface perpendicular to the osteonal axis was imaged for each sample with a Quanta 250 FEG microscope (FEI, Hillsboro, Oregon, USA) equipped with a GDA detector in environmental conditions. Thus, sample preparation was not required. The microscope was operated at a voltage of 30 kV and hydrostatic pressure of 0.9 Torr (120 Pa) in the chamber. To reflect the difference in mineralization (Roschger et al., 1998, 2008; Fratzl-Zelman et al., 2009), acquisitions were performed in the backscattered electron mode. A magnification of 50 and a very slow scanning (30 ms per point) were used to obtain an image of the maximum surface with an optimal contrast. The resulting images were in 24 bit TIF format with a resolution of 4096 3536 pixel. For each sample, the number of osteocyte lacunae was counted manually from the SEM images with a superimposed grid (Fig. 1). Based on these images, thresholding was performed manually with the free software imageJ to separate the tissue (in white) from the porosity in order to have the macroscopic porosity in black (Fig. 1). The tissue surface was defined as the image area minus the porosity area, and the osteocyte lacunar density (#/mm2) was calculated as the ratio of the lacunae number over the tissue area.
Compression testing
Each hydrated sample was tested in a homemade compression machine that was controlled by a LabView acquisition card (National Instrument Corporation, Austin, Texas, US). The sample was placed between two cylindrical jaws and maintained by a 10 N pre-load. A displacement rate of 0.7 mm/ s was applied to the mobile jaw. The test consisted of three steps; first, the sample was loaded until 100 N, and then the sample was unloaded and loaded again to fracture. The load and displacement were registered during the test by means of an extensometer and a 2.5 kN load cell. The stress-strain curve was derived from the load-displacement curve by normalizing the load by the initial section and the displacement by the initial length. The macroscopic Young’s modulus (E) was defined as the slope of the linear unloading portion. The inflection point at the boundary between the linear and non-linear domains was defined as the yield stress (σy). The ultimate stress (σmax) was defined as the maximum stress reached during the test, and the ultimate strain was the strain associated with this stress.
2.4. Scanning electron microscopy and osteocyte lacunae counting Scanning electron microscopy (SEM) was used to image the sample after compression testing. In the present study, a
Fig. 1 – (a) SEM image of a OI sample (OI 4) with a superimposed grid (b) binary image made via thresholding from (a) without grid.
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2.5.
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Statistical analysis
A statistical analysis was performed using R (The R foundation) with a threshold of 5% corresponding to the alpha risk. A nonparametric Mann–Whitney test was used to detect any significant differences between the parameters of the pathological bones and the controls. The influence of the porosity on the mechanical properties was studied using a Spearman correlation.
3.
Results
3.1.
Microstructure observations
Two scales of porosity were observed on these figures: (i) the vascular intracortical porosity, which corresponds to the Haversian canals in healthy bones and present larger cavities in OI samples, and (ii) the osteocyte lacunar porosity, with a higher number of lacunae in OI bone. To quantify this effect, the osteocyte lacunar density (number of osteocyte lacunae per area) was measured. The density was significantly higher for OI samples (6407195.5 #/mm2) compared with controls (395.8715.8 #/mm2, p¼ 0.024) (Fig. 5). Based on the SEM images of samples after the compression tests, the crack paths also differed between OI and control bones. Based on observations, a major crack appeared on all control samples, whereas smaller but more numerous cracks were observed on the OI samples surface.
Scanning electron microscopy in backscattered electron mode was used to image the samples after compression testing. Figs. 2–4 show the microstructures of cortical bones for healthy and diseased children.
Fig. 2 – SEM images of the three controls (C1, C2, C3 from top to bottom) taken after compression test.
Fig. 3 – SEM images of three OI samples (OI 1, OI 2, OI 3 from top to bottom) taken after compression test.
journal of the mechanical behavior of biomedical materials 46 (2015) 261 –270
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Fig. 4 – SEM images of two OI samples (OI 5, OI 6 from top to bottom) taken after compression test.
Fig. 6 – 3D images of (a) a control and (b) a OI reconstructed from the tomography acquisitions. The left part of each sample is inverted to show only the porosity.
Fig. 5 – Comparison of osteocyte lacunar density between the control group and the OI group (npo0.05).
3.2.
Vascular intracortical porosity
The vascular intracortical porosity was measured using high resolution X-ray computed tomography. The difference in the vascular intracortical porosity was observed on 3D images reconstructed from the tomography acquisitions, for which one part of the sample was inverted to reveal the porosity. Fig. 6 shows the typical 3D images of control and OI samples reconstructed from the tomography acquisitions. The cavities visible on the surface
on SEM images were present in the entire structure. The porosity microarchitecture, which consisted of thin channels in control bone, was replaced by a rougher architecture in OI bone. The values of the vascular intracortical porosity for each sample are given in Table 2. As expected, the porosity was significantly higher in OI samples compared with controls.
3.3. Bone mineral density (BMD) and tissue mineral density (TMD) The bone mineral density (the density in mineral (hydroxyapatite HA) of the entire sample including the porosity) was measured based on the tomography acquisitions. The results are given in Table 2. The BMD of OI bones was significantly lower than that of control bones. On the contrary, the TMD was significantly higher in OI samples than in controls. The porosity and BMD were significantly negatively correlated (r2 ¼0.89, p¼ 0.017).
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journal of the mechanical behavior of biomedical materials 46 (2015) 261 –270
Table 2 – Values of structural parameters (BMD, porosity and TMD) and mechanical parameters (compressive Young’s modulus, ultimate strength, ultimate strain and yield strength) for each sample. Samples
BMD (gHA/cm3)
Porosity (%)
TMD (gHA/cm3)
E (GPa)
σmax (MPa)
σy (MPa)
OI 1 OI 2 OI 3 OI 4 OI 5 OI 6 Mean SD Control 1 Control 2 Control 3 Mean SD
0.621 0.859 0.771 0.909 0.854 0.901 0.819a 0.109 0.918 0.941 0.998 0.952 0.041
45.2 16.6 33 16.5 24.6 16.7 25.4a 11.7 6 2.7 2.2 3.6 2.1
1.133 1.030 1.151 1.089 1.133 1.082 1.103a 0.045 0.977 0.967 1.020 0.988 0.028
3.9 3.3 4 4.7 2.5 5.5 4.0a 1.0 10.5 9.6 6.8 9.0 1.9
49.1 78.6 58.9 87.9 51.7 79 67.5a 16.3 114.2 163.9 151.9 143.3 25.9
33 57 49 70 38 60 51a 14 90 105 80 92 13
BMD¼ bone mineral density, TMD ¼ tissue mineral density. E ¼ Young’s modulus, σmax ¼ultimate stress, and σy ¼yield stress. a Significant difference with the mean values of the control population with a threshold of 5%.
Fig. 7 – Typical stress strain curve for a OI sample.
3.4.
Macroscopic mechanical properties
A typical stress strain curve is presented in Fig. 7, there is a first loading part, then a linear unloading part used to derive the Young’s modulus. Besides, after the maximum stress the stress decreased progressively due to microcracks formation. The values of Young’s modulus, yield stress and ultimate stress, which were derived from the stress–strain curves obtained during the compression tests, are presented in Table 2. These three mechanical parameters were significantly lower in OI samples than in controls. The yield stress and ultimate stress linearly correlated with porosity in OI samples (r2 ¼0.78, p¼ 0.33 for both) (Table 3). Similarly, the yield stress and ultimate stress linearly correlated with BMD (r2 ¼ 0.89, p¼ 0.017 for both) with a slope opposite to the slope of the correlation obtained for the porosity. Furthermore, the Young’s modulus and the microstructural parameters did not correlate, i.e., BMD, TMD, or porosity. Similarly, the
ultimate strain, which corresponds to the strain measured at the ultimate stress point, did not correlate with these microstructural parameters. Notably, none of these correlations were significant for the control group due to the low number of controls; however, the trend between the ultimate stress and porosity was the same with a higher slope.
4.
Discussion and conclusions
In the present study, the mechanical properties and microstructural parameters of bone tissue from patients with OI and controls were analyzed at the macroscopic (sample) level with compression testing, high-resolution X-ray computed tomography and scanning electron microscopy. All samples retrieved from healthy children or patients with OI originated from the long bones, irrespective of the gender distribution. Due to the difficulty in retrieving bone samples from children
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Table 3 – Correlations between the mechanical properties and the microstructure parameters within the OI group. Mechanical properties
Microstructure parameters
Young’s modulus
Vascular intracortical porosity BMD TMD Osteocyte lacunar density
Yield stress
Ultimate stress
R
p-Value
0.02 2.12 3.82 0.0001
0.257 0.486 0.145 0.086
0.65 0.35 0.78 0.91
Vascular intracortical porosity BMD TMD Osteocyte lacunar density
0.9687 99.614 191.53 0.0118
0.886 0.943 0.580 0.029
0.033 0.016 0.22 1
Vascular intracortical porosity BMD TMD Osteocyte lacunar density
1.17 113.7 276.6 0.0026
0.886 0.943 0.580 0.029
0.03 0.016 0.22 1
and the scarcity of the pathology, the number of samples remained low. In the present study, all patients with OI were treated with bisphosphonate (BP) therapy. Although the pathological and healthy groups differed in two ways, our results were clinically relevant because BPs are a common treatment for OI. BPs have been proven to prevent fractures and increase mineral density (Glorieux, 2008; Cundy, 2012; Vyskočil et al., 2005; Shapiro et al., 2003). Boyde et al. (1999), Roschger et al. (2008) showed that the mineral density increased in OI patients without BPs; thus, the trend in the tissue mineral density observed in our study was correct, and the effect of the pathology was simply overestimated. Similarly, BPs decreased the cortical porosity (Shapiro et al., 2003; Roschger et al., 2001), and the trend observed in the present study was correct with underestimated values. To the best of our knowledge, only one very recent study performed macroscopic mechanical tests on bones from children treated with BPs and untreated controls to assess the effects of BPs on the mechanical properties of OI. In this study, Albert et al. (2014) did not draw conclusions about the effects of BPs on the mechanical properties because they only examined two nontreated samples and the strength of one sample fell within the range of treated samples. In a previous study, we investigated the tissue level and we found that the mechanical properties were lower in OI samples treated with bisphosphonates compared to control samples. Besides, the tissue mineral density was higher in OI samples, correlated to the tissue Young’s modulus measured by nanoindentation, unlike the crystallinity (Imbert et al., 2014). Bisphosphonates might have an effect on the mineral nanostructure and the mechanical properties measured by nanoindentation on osteoporotic bones (Bala et al., 2012) but no similar results on OI patients were found. The effects of BPs on OI bones and on osteoporotic bones are likely to be different. The microstructures observed with SEM using the backscattered electron mode differed between healthy and pathological bones. In particular, the osteocyte lacunar density was calculated and found to be significantly higher in OI bones compared with control bones. Many authors have been interested in osteocyte lacunar density in OI children (Albert et al., 2014), healthy animals (Nicolella et al., 2006; Ma et al., 2008) or healthy human adults (Qiu et al., 2005;
Slope
Vashishth et al., 2000; Dong et al., 2010; Carter et al., 2013). The osteocyte lacunae correspond to osteoblasts embedded in the matrix, therefore their number is linked to osteoblast activity. The increased number of osteocyte lacunae is in agreement with an increased number of osteoblast described in the paper of Rauch et al. (2000). Moreover values found in the present study were on the same order of magnitude as results reported in the literature. Others have qualitatively observed a difference in the osteocyte lacunar density in human OI bones (Rauch et al., 2000; Jones et al., 1999) without direct comparison to control bones. To our knowledge, the only quantitative 3D analysis that compared OI and controls was performed for a mouse model (Carriero et al., 2014). As in the present study, the results showed a significant increase in the osteocyte lacunar density for oim compared with wildtype mice (127 365 vs. 72 981 #/mm3) (Carriero et al., 2014). Moreover, larger and more numerous cavities were observed in OI bones. In our study, these large cavities were indicated by the vascular intracortical porosity, which was measured by high-resolution computed tomography and significantly higher in OI samples compared with controls. To date and to our knowledge, the data on cortical porosity in human OI bone are scarce. Albert et al. (2014) measured the vascular intracortical porosity of nine OI donors and found values of 21710%, which agreed with our results. Pazzaglia et al. obtained a higher fraction of the vascular/resorption area in children with OI (between three and eight years) compared with controls (44.3% vs. 13.6%) (Glorieux, 2008). In the oim study, the vascular porosity was measured by tomography (Coleman et al., 2012; Carriero et al., 2014), and Carriero et al. (2014) recently did not find any significant difference in the cortical porosity between oim and wild-type mice (7.8 (2.7) vs. 6.1 (4.6)). A combination of a higher turnover (Cepollaro et al., 1999; Rauch et al., 2000), a slower bone formation due to fewer efficient osteoblasts (Rauch et al., 2000; Roschger et al., 2008) and higher resorption parameters (Rauch et al., 2000; Roschger et al., 2008) may lead to resorption that is not followed by formation, which may explain the formation of these cavities. This modification in the bone remodeling process leading to a modification of osteonal microstructure could not be transposed to the homogeneous tissue of mice. Moreover, the difference in the vascular and lacunar porosities could explain the shorter cracks observed in OI. The cracks did not propagate as well as in controls
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because they met cavities more frequently during propagation. Ma et al. (2008) already reported a negative correlation between the osteocyte density and micro-crack length in rats, suggesting that lacunae are able to stop or initiate cracks in order to dissipate energy. The increased lacunar density might be a way to compensate for the material fragility. Other authors observed a relationship between the osteocyte lacunar density and the micro-crack density (Vashishth et al., 2000) but could not conclude the cause. We hypothesized that the poor mineral quality (Imbert et al., 2014) increased the crack density, and these cracks caused the death of some osteocytes by disrupting the supply of nutrition to these cells, which then increased bone remodeling turnover. Knowledge of the living osteocyte density compared with the osteocyte lacunar density should allow the validation of this assumption. As mentioned by others (Reinus et al., 1998), an increase in the porosity led to a decrease in bone mineral density (BMD). As expected, the BMD measured by high-resolution computed tomography was significantly lower in OI bones compared with controls. At the tissue scale, the tissue mineral density, derived from BMD and porosity, was significantly higher in OI bones compared with controls. This result agreed with previous studies showing that OI bone was more mineralized than normal bone in humans (Roschger et al., 2008; Boyde et al., 1999) and mice (Vanleene et al., 2012; Grabner et al., 2001). The macroscopic Young’s modulus, yield stress and ultimate stress were measured during compression tests and were significantly lower in OI subjects compared with controls. In the literature, the mechanical properties of bones from patients with OI were mainly measured at the tissue scale by nanoindentation (Imbert et al., 2014; Albert et al., 2013; Fan et al., 2007), but few mechanical data at the sample scale exist, and direct comparisons of the mechanical properties at this sample scale between OI and healthy children have not been performed. In the present study, the results obtained for the bones of healthy children were consistent with results obtained in several previous works (Berteau et al., 2014; Öhman et al., 2011). Albert et al. recently performed three-point bending tests on OI cortical bones that required linear elastic beam theory to extract the mechanical parameters. They calculated mean values of 1.670.4 GPa, 20.876 MPa and 26.578.6 MPa for the Young’s modulus, the yield Stress and the ultimate stress, respectively, in the transverse direction (Albert et al., 2014). These results are clearly weaker than the results obtained from compression tests in the present study. The difference could be explained by the nature of the three-point bending test; the stress field is not uniform and mixes the tensile and compression area. The yield and ultimate stresses are strongly sensitive to these two types of loading (Bayraktar et al., 2004). Three-point bending data have also been reported for the oim model (Vanleene et al., 2012, 2011; Yao et al., 2013). However, Vanleene et al. (2011) did not measure a significant difference in the Young’s modulus between oim and wild-type mice (7.071.2 GPa vs. 6.970.7 GPa). Their values were higher than the values measured in humans, proving again that the macrostructure should be taken into account for human samples.
In this study, we aimed to investigate the relationships between the mechanical properties and the microstructure parameters within the OI population in order to identify the factors responsible for OI bone fragility. We found a strong linear correlation between the stresses (yield stress and ultimate stress) and the microstructural parameters, such as the bone mineral density and porosity. The BMD, which takes into account the porosity and tissue mineral, density explained 89% of yield and ultimate stresses, while porosity alone explained 78% of these mechanical parameters. These results showed that the tissue quality was not the only factor responsible for the fragility and that porosity played a significant role. In the literature some studies investigated the microstructure–mechanical properties relationship in bovine (Li et al., 2013) or human (Ks and Nicolella, 2012) bones but, to our knowledge, only one study (Albert et al., 2014) has attempted to identify correlations in OI bones and found no significant correlation between the mechanical properties calculated in the transversal direction and the microstructural parameters. The effect of porosity on the mechanical properties was also investigated for the oim model. For example, Carriero et al. (2014) showed that regions prone to fracture were located mostly around canals. In conclusion, this study measured the structural and mechanical parameters of cortical bone samples from children with OI and compared these values to those measured in controls. The results showed that the mechanical properties were significantly lower in OI bones, while the porosity was significantly higher. The correlation between both was strong but limited, which proved the influence of the tissue quality. The osteocyte lacunar density was significantly higher in OI bones, suggesting that remodeling was altered in OI bones. Although the mechanical properties were found to negatively correlate with the porosity, we did not find a correlation between the osteocyte lacunar density and the mechanical properties. Further investigations from a biological perspective are required to establish a relationship between the osteocyte lacunar density and the bone fragility.
Acknowledgments The authors are grateful to G. Finidori and S. Avril for their helpful discussion. We would also like to thank the engineers and technicians B. Ponsard, O. Pollet, C. Bosser and B. Jeanpierre for their technical support. We also wish to thank the Rhône-Alpes Region, the IVTV ANR-10-EQPX-06-01, ANR OMBIOS and the AOI (Association de l’Ostéogénèse Imparfaite) (72 ANR OMBIOS 11-BS09-036-01) for their financial support.
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