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The role of mineralization and organic matrix in the microhardness of bone tissue from controls and osteoporotic patients
Bone 43 (2008) 532–538
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Bone j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b o n e
The role of mineralization and organic matrix in the microhardness of bone tissue from controls and osteoporotic patients G. Boivin a,⁎, Y. Bala a, A. Doublier a, D. Farlay a, L.G. Ste-Marie b, P.J. Meunier a, P.D. Delmas a a b
INSERM Unité 831, Université de Lyon, Faculté de Médecine R. Laennec, Lyon, France Centre de Recherche du CHUM, Hôpital Saint-Luc, Université de Montréal, Montréal, Canada
a r t i c l e
i n f o
Article history: Received 28 January 2008 Revised 27 May 2008 Accepted 28 May 2008 Available online 10 June 2008 Edited by: Rene Rizzoli Keywords: Microhardness Bone mineralization Osteoporosis Vickers indentation Quantitative microradiography
a b s t r a c t Degree of mineralization of bone (DMB) is a major intrinsic determinant of bone strength at the tissue level but its contribution to the microhardness (Vickers indentation) at the intermediary level of organization of bone tissue, i.e., Bone Structural Units (BSUs), has never been assessed. The purpose of this study was to analyze the relationship between the microhardness, the DMB and the organic matrix, measured in BSUs from human iliac bone biopsies. Iliac bone samples from controls and osteoporotic patients (men and women), embedded in methyl methacrylate, were used. Using a Vickers indenter, microhardness (kg/mm2) was measured, either globally on surfaced blocks or focally on 100 μm-thick sections from bone samples (load of 25 g applied during 10 sec; CV = 5%). The Vickers indenter was more suited than the Knoop indenter for a tissue like bone in which components are diversely oriented. Quantitative microradiography performed on 100 μm-thick sections, allowed measurement of parameters reflecting the DMB (g/cm3). Assessed on the whole bone sample, both microhardness and DMB were significantly lower (−10% and −7%, respectively) in osteoporotic patients versus controls (p b 0.001). When measured separately at the BSU level, there were significant positive correlations between microhardness and DMB in controls (r2 = 0.36, p b 0.0001) and osteoporotic patients (r2 = 0.43, p b 0.0001). Mineralization is an important determinant of the microhardness, but did not explain all of its variance. To highlight the role of the organic matrix in bone quality, microhardness of both osteoid and adjacent calcified matrix were measured in iliac samples from subjects with osteomalacia. Microhardness of organic matrix is 3-fold lower than the microhardness of calcified tissue. In human calcanei, microhardness was significantly correlated with DMB (r2 = 0.33, p = 0.02) and apparent Young’s modulus (r2 = 0.26, p = 0.03). In conclusion, bone microhardness measured by Vickers indentation is an interesting methodology for the evaluation of bone strength and its determinants at the BSU level. Bone microhardness is linked to Young’s modulus of bone and is strongly correlated to mineralization, but the organic matrix accounts for about one third of its variance. © 2008 Elsevier Inc. All rights reserved.
Introduction Bone strength is influenced by a number of different determinants, in addition to mass, size, geometry and microarchitecture, bone strength is also influenced by the intrinsic material properties of the tissue. These include the mineralization of bone and the characteristics of the organic matrix (orientation and chemical structure of the collagen fibers), the accumulation of microdamage, and indirectly the apoptosis of osteocytes [1–4]. Bone “quality” is profoundly influenced by the rate of bone turnover [5]. The major role of mineralization has been emphasized by the fact that fracture risk and bone mineral density were changed in osteoporotic patients treated with antiresorptive agents, without modifications of bone
⁎ Corresponding author. INSERM Unité 831, Université de Lyon, Faculté de Médecine R. Laennec, 69372 Lyon Cedex 08, France. Fax: +33 4 78 77 86 63. E-mail address: [email protected] (G. Boivin). 8756-3282/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.05.024
matrix volume or bone microarchitecture. This was largely, if not totally, due to modifications of the duration of secondary mineralization [6–13]. The degree of mineralization of bone (DMB) not only influences the mechanical resistance of bone [14] but also partly determines the bone mineral density [6]. From microradiographic observations [15– 17], it is clear that DMB varies over bone structural units (BSUs, namely the osteons in cortical bone and the trabecular packets in cancellous bone), with the recently deposited ones being much less mineralized than the older ones (Fig. 1). This heterogeneity in the DMB is explained by the fact that bone formation, which follows bone resorption in the remodeling sequence is a multi-step process. Following its deposition, the new matrix begins to mineralize after about 5 to 10 days from the time of deposition, and the linear rate of this primary mineralization can be measured directly in vivo using double tetracycline labeling. After full completion of the BSUs, a phase of secondary mineralization begins [16–18]. This process consists of a slow and gradual maturation of the mineral component, including an
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Fig. 1. Human bone samples. Microradiograph of a 100 ± 1 μm-thick section illustrating the heterogeneity of the mineralization in the various BSUs (young bone) and in the interstitial old bone tissue (left). The brighter interstitial lamellae had a mineral content of about 1.40 g/cm3 and a hardness of about 65 kg/mm2 while the least highly mineralized BSUs had a mineral content of about 1.00 g/cm3 and a hardness of less than 40 kg/mm2. Unstained sections of cortical (upper right) and trabecular (lower right) with the Vickers impressions into bone tissue.
increase in the amount of crystals, an augmentation of crystal size toward their maximum dimensions and an increase in the perfection of the crystals [19,20]. If remodeling activity is not accelerated, this secondary mineralization progressively augments the mineral content of bone matrix, until the maximum mineral content is reached. Unknown in humans, the duration of the secondary mineralization has been reported in rabbits [21] and is now described in an animal model (ewe) having a Haversian remodeling similar to humans [22]. Quantitative microradiography giving a high linear resolution, allowed measurement of the focal DMB of each BSU within the limits imposed by the thickness of the section [15,18,23]. Variations of mineral density have also been evaluated in cancellous bone by others through quantitative backscattered electron imaging [16,24] and, in total bone, by synchrotron radiation microtomography [10,12]. Hardness is a measurement used to assess the resistance of a material deformation, that is to say, the ability of a material's surface to resist indentation with or without permanent indentation [25–27]. Microhardness tests allow the investigation of relationships between the structure and the mechanical characteristics of a material at a microstructural scale. In various bones, microhardness, mineral content and Young’s modulus increase with age [28]. The microhardness test is a valuable tool by which our understanding of the mechanics of bone can be improved. The dimensions of an impression that results from forcing an indenter into the surface of a material, with a known and constant load, lead to the calculation of the hardness value. The pyramidal square-based Vickers indenter has been often used for indenting bone (Fig. 1). It has been suggested that the Knoop indenter has an advantage over the Vickers one (see below) for analyzing cortical bone microhardness [29]. In contrast to nanoindentation for assessing parameters at the lamellar level [30], microhardness measured by microindentation appears most suited to test mechanical properties of bone at the intermediary level of organization of bone, i.e., BSUs. The mineral content of bone is correlated with Young's modulus in cortical bone [31], and microhardness values increase with Young's modulus [32–34]. However, no extensive and comparative analysis of microhardness and mineralization of bone tissue has been done in bone samples. Early studies only
reported partial data (microhardness calculated in different kinds of osteons based on the grey-levels in microradiographs) [35,36]. We hypothesized that a strong relationship exists between microhardness and mineralization. The main purposes of the present study were to investigate parameters reflecting microhardness and mineralization at the BSU level in controls and osteoporotic patients (men and women), and to test the potential relationship between these parameters. The role of organic matrix in the microhardness of bone was also analyzed. Materials and methods Subjects and bone samples We obtained: - iliac bone samples taken at necropsy from subjects who died suddenly without apparent bone disease, representing the control group, 6 women (82 ± 7 years) and 13 men (66 ± 4 years), - iliac bone biopsies from 30 men (48 ± 11 years) suffering from untreated idiopathic osteoporosis [13 from France (50 ± 11 years) and 17 from Canada (49 ± 8 years)], osteoporotic patients have been diagnosed on clinical, radiological, biological and histomorphometric criteria, - iliac bone biopsies taken from 22 untreated women with postmenopausal osteoporosis (62 ± 5 years) corresponding to a subset of a large group [8], - calcanei excised post-mortem from 18 subjects (78 ± 8 years) and previously used for measurement of Young's modulus and maximal strength in compression tests [14], - iliac bone biopsies from 13 patients (47 ± 11 years) suffering from untreated osteomalacia diagnosed on clinical, radiological, biological and histomorphometric criteria. Iliac bone samples were fixed in 70% alcohol, dehydrated in absolute alcohol, then embedded in methyl methacrylate without prior decalcification [37]. For quantitative microradiography, thick sections (about 150 μm) were cut from embedded bone samples with
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a precision diamond wire saw (Well, Escil, Chassieu, France), progressively ground to a thickness of 100 μm, and polished with a diamond paste (1 μm). The thickness of the section was measured with an accuracy of 1 μm using a precision micrometer (Compac, Geneva, Switzerland). After ultrasonic cleaning the bone sections were microradiographed [15,16]. For microhardness tests, either the embedded blocks or the 100 μm-thick sections previously used for microradiography, were meticulously surfaced and polished with an alumina suspension (1 μm). If orientation of the blocks was possible before sectioning, the cutting plane perpendicular to the Haversian canals of cortical bone was preferred. Quantitative microradiography Contact microradiography of 100 μm-thick bone sections was performed using a X-ray diffraction unit PW 1830/40 equipped with a diffraction tube PW 2273/20 (Philips, Limeil Brévannes, France) operating at 25 kV and 25 mA. The nickel-filtered copper Kα radiation was used. A Geola high-resolution film (VRP-M green sensitive) was exposed for 20 min (Slavich International Wholesale Office, Vilnius, Lithuania). For quantitative evaluation of the X-ray absorption by the bone section, aluminum step-wedges were exposed on each microradiograph [15]. The DMB was quantified using a combined contact microradiography, microdensitometry computerized method [15]. This procedure used new automatic programs for analyzing grayness levels (MorphoExpert and Mineralization, ExploraNova, La Rochelle, France). A digital camera (actual resolution: 1600 × 1200 pixels or 800 × 600 after binning) captures the microscopic image of the microradiograph. After calibration using the aluminum reference system, the measured regions of bone tissue are automatically selected, then the gray levels are segmented after bone thresholding. The values of the gray levels are then obtained at pixel level (for a magnification ×2.5, the size of the pixel is 2.82 μm). Finally, gray-level values are converted into DMB values with the construction of a calibration curve based on the measurements obtained on the aluminum step-wedge. DMB are finally expressed in g of mineral over cm3 of bone (g/cm3) and are measured separately in cortical, cancellous, and total (cortical + cancellous) bone tissues. For cortical bone, it represents the mean of the DMB measured separately in the thickest and the thinnest cortices. The main parameters, extracted from DMB measurements, were the mean DMB, the mean highest and most frequent DMB (DMB Freq. Max.) and the mean index of heterogeneity of the distribution of DMB expressed as the mean of the widths at half-maximum measured on the individual DMB curves. Measurement of microhardness Microhardness is measured using a Micromet 5104 (Buehler, Lake Bluff, Illinois, USA) equipped with a Vickers indenter which is more suitable than the Knoop indenter [38]. The microhardness tester is linked to a computer with an OmniMet program allowing the calculation of microhardness and then the processing of the various values measured. The sizes of the impressions are adapted to the study of the intermediary level of organization of bone, i.e. the BSU level. Nanoindentation hardness testing is now used frequently with load forces less than 1 g being applied. However, this does require more stringent environmental control of factors such as vibration, airflow, and temperature. Also, the indentations are typically very small, in bone tissue they are at the level of the lamellae which are not a level of investigation for bone remodeling [27,39,40]. Iliac bone samples and sections are adapted in size and shape to the specimen holders used, and a good surface condition is required to enhance the precision of hardness evaluation. Due to the relatively small size of the impressions created during microhardness tests and because the BSU values were very homogeneous, we were able to take the average of 2 to 5 impressions from any BSU to
give the value of microhardness for that BSU (Fig. 1). In human bone tissue, all the measurements of microhardness are constant in a BSU, but the microhardness differs from one BSU to another in agreement with the wide heterogeneity of the secondary mineralization. The formulae used to calculate the microhardness of the tissue are: for Knoop tests, Hk = 13689.9 P/d2 (where Hk is Knoop microhardness expressed in kg/mm2, P is test load expressed in g, d is length of the longer diagonal expressed in mm), for Vickers tests, Hv = 1854.4 P/d2 (where Hv is Vickers microhardness expressed in kg/mm2, P is test load expressed in g, d is the mean length of the two diagonals expressed in mm). In our laboratory, microhardness tests with Vickers indenter are performed at a load of 25 g for 10 s. This choice was made after testing in the same bone samples, loads of 10, 25, 50 g applied for 5, 10 and 15 s. A combination of 25 g for 10 s was finally chosen for the tests at the surface of blocks and of bone sections. Indeed, the depth of the impression is always inferior to 10 μm. Hence, there was no risk that the impression pierced the section, allowing the hardness of the specimen holder to influence the values. Similar values (load) have been recently applied to bone samples [41,42], and the intraand inter-observer coefficients of variation were ≤5%. In control specimens in which the amount of bone available was larger than in a bone biopsy, faces cut perpendicularly or in parallel to Haversian canals were both used for the estimation of the influence of the orientation of the tissue on the microhardness measurements. For this purpose, a comparison of the values obtained in the same BSUs with Vickers and Knoop indenters was performed. In fact, in each BSU tested, hardness values have been measured with Vickers indenter without particular orientation, with Knoop indenter having their longest diagonal oriented first parallel and second perpendicular to the main arrangement of the lamellae (Fig. 2). Vickers microhardness corresponded to the mean microhardness measured on Knoop impressions oriented perpendicularly or in parallel to bone lamellae. Since no single microhardness measurement can precisely reflect the microhardness of bone tissue as a whole, a study was performed to evaluate the optimum number of impressions required to obtain a correct and reproducible value of the microhardness on the surfaced blocks. Up to 120 different measurements were performed for each sample showing that the mean microhardness did not change after 60 measurements. For example, in control subjects, cortical Hv was 48.84 ± 3.25 kg/mm2 for 100 impressions and 48.83 ± 3.27 kg/mm2 for 40 indentations. Thus, global microhardness calculated at the level of the surface of a bone sample is reported as the mean value of 60 individual
Fig. 2. Bone sample from a control woman with both Vickers and Knoop impressions. In 6 control women, mean Vickers microhardness corresponded to the mean values from Knoop impressions oriented perpendicular and parallel to bone lamellae.
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Table 1 Human control and osteoporotic (OP) patients Human subjects
19 Controls 52 OP patients 30 OP men 22 OP women 13 control men 6 control women
Total bone
Cortical bone
Cancellous bone
Hv25 (kg/mm2)
DMB (g/cm3)
HI (g/cm3)
Hv25 (kg/mm2)
DMB (g/cm3)
HI (g/cm3)
Hv25 (kg/mm2)
DMB (g/cm3)
HI (g/cm3)
49.18 ± 1.82 44.10 ± 4.72⁎ 40.52 ± 1.88# 48.97 ± 2.46 48.96 ± 1.85 49.71 ± 2.01
1.10 ± 0.09 1.03 ± 0.07⁎ 1.02 ± 0.06# 1.05 ± 0.08 1.10 ± 0.09 1.10 ± 0.08
0.22 ± 0.07 0.25 ± 0.07 0.23 ± 0.06 0.28 ± 0.07 0.22 ± 0.08 0.21 ± 0.03
49.30 ± 2.16 44.05 ± 4.69⁎ 40.59 ± 2.07# 48.77 ± 2.66 49.02 ± 2.03 49.91 ± 2.51
1.10 ± 0.09 1.01 ± 0.07⁎ 1.00 ± 0.06# 1.03 ± 0.08 1.10 ± 0.09 1.09 ± 0.08
0.21 ± 0.07 0.24 ± 0.06 0.22 ± 0.05 0.25 ± 0.06 0.21 ± 0.08 0.21 ± 0.04
48.92 ± 1.57 44.23 ± 5.07⁎ 40.28 ± 2.15# 49.30 ± 2.54 48.73 ± 1.62 49.33 ± 1.52
1.11 ± 0.08 1.07 ± 0.08⁎ 1.08 ± 0.09# 1.08 ± 0.08 1.11 ± 0.06 1.12 ± 0.10
0.19 ± 0.05 0.23 ± 0.06⁎ 0.21 ± 0.04 0.24 ± 0.07 0.19 ± 0.06 0.20 ± 0.03
Mean values (±SD) for the Vickers microhardness (Hv25), the degree of mineralization of bone (DMB) and the heterogeneity index of the distribution of DMB (HI). *pb0.001 and epb0.003 versus corresponding controls.
measurements (20 for each cortex and 20 in the cancellous bone), taken at random but separated by at least 500 μm. The distance between impression and the boundary of bone is at least 10 μm. Because the BSU is the intermediary level allowing evaluation of the changes due to remodeling activity, focal microhardness was measured on the 100 μm-thick sections used for microradiography, in order to identify the BSUs according to their DMB (Fig. 1). Newly formed bone (recent BSUs with a low DMB on microradiography) and old bone (mainly interstitial with high to medium DMB), were tested both in cortical and cancellous bone tissues. Finally, the correlation between both microhardness and DMB was tested at the BSU level, to assess the role of mineralization in the hardness of the tissue. Statistical analysis Results were expressed as mean ± standard deviation (SD). According to the number of samples in the groups, parametric (unpaired Student’s t test for the comparison of means) and non-parametric (Mann–Whitney’s U test for the comparison of means, Spearman test for the correlations between two parameters) tests were used. Significant results were accepted when p ≤ 0.05. Results Parameters reflecting the mineralization of bone and its microhardness were measured in iliac bone samples from control men and women, and men and women with idiopathic osteoporosis. DMB and microhardness values were significantly lower in osteoporotic patients compared to controls (Table 1). The decreases (−7%) were observed in both in men and women but the decrease was significant only for men (Table 1). DMB and microhardness did not change with age in controls (p N 0.05 r2 = 0.010 and 0.002, respectively) and
Fig. 3. Measured separately in 185 BSUs from osteoporotic women (black dot, y = 33.31x + 14.87) and 80 BSUs from osteoporotic men (open square, y = 32.71x + 14.33), Vickers microhardness was significantly (p b 0.0001) correlated with the degree of mineralization of bone.
untreated osteoporotic patients (p = 0.76, r2 = 0.003, and p = 0.36, r2 = 0.03, respectively). The highest and most frequent DMB was significantly decreased (−10%, p b 0.003) in osteoporotic patients (1.00 ± 0.09 g/cm3) compared to controls (1.10 ± 0.10 g/cm3). Even if a tendency to the augmentation of the heterogeneity of the mineralization is noted (p = 0.064 in total bone, Table 1), the mean heterogeneity index was not significantly modified. All parameters (mineralization and microhardness) analyzed were similar in the two populations (French and Canadian) of osteoporotic men and were given for the global group of osteoporotic men. Microhardness and parameters reflecting the secondary mineralization of bone were not significantly different when measured separately in cortical and cancellous bone tissues (Table 1). No difference was observed in the two cortices, the thickest and the thinnest (data not shown). Microhardness was lower in recent BSUs than in old interstitial tissue as quantified by focal measurements at the BSU level. When measured separately at BSU level, there were significant positive correlations between microhardness and DMB in controls (r2 = 0.36, p b 0.0001), osteoporotic men (r2 = 0.52, p b 0.0001, Fig. 3) and osteoporotic women (r2 = 0.40, p b 0.0001, Fig. 3). If these two groups of osteoporotic patients were analyzed together representing measurement of 265 BSUs, the correlation was also significant (r2 = 0.43, p b 0.0001). Thus, the level of secondary mineralization appears to be the major cause of change in the microhardness of bone tissue, but mineralization explains only 40 to 50% of the variance of the microhardness of bone tissue. However, for a given value of DMB (Fig. 3), a large variation in microhardness was observed. Conversely, for a given value of microhardness, various DMB values were found. This variation was emphasized by the observation of the individual curves in osteoporotic men (Fig. 4) and women showing similar slopes despite different spectra of DMB.
Fig. 4. In 8 osteoporotic men, there are significant correlations between the Vickers microhardness and the degree of mineralization of bone within subjects, but large between subjects variability (from r2 = 0.80 to 0.76, the regressions equations are the following: y = 0.018x + 0.072, y = 0.019x + 0.058, y = 0.014x + 0.304, y = 0.013x + 0.349, y = 0.022x − 0.046, y = 0.014x + 0.366, y = 0.012x + 0.510, y = 0.022x + 0.133). Similar observations (not shown) were obtained in osteoporotic women.
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Fig. 5. Stained section of bone from a patient with osteomalacia showing both calcified and osteoid tissues (right). Vickers microhardness (Hv), separately measured in calcified matrix (upper left) and in osteoid tissue alone (lower left) in patients with osteomalacia (n = 13), illustrates the role of osteoid tissue that accounted for 36.4 ± 2.7% of microhardness of calcified matrix.
To highligtht the role of organic matrix in bone quality, microhardness of both osteoid and adjacent areas of calcified matrix were measured in iliac bone samples from patients with osteomalacia. Microhardness of osteoid tissue is about 3-fold lower than the total microhardness of calcified matrix located in the vicinity of non mineralized matrix (Fig. 5). Furthermore, this observation was reinforced by the fact that in osteoporotic patients, regressions show that, if a value 0 is attributed to DMB, the microhardness values would be 15.66 and 14.33 kg/mm2 in women and men, respectively (Fig. 3) and 14.85 kg/mm2 in the whole group of osteoporotic patients. These values match closely to the mean value of microhardness measured in osteoid tissue in biopsies from patients diagnosed with osteomalacia (Fig. 5). We analyzed the correlations between mineralization, microhardness measured at the tissue level and biomechanical properties previously measured at organ level in contiguous samples taken from human calcanei mainly constituted of cancellous bone. Microhardness was significantly correlated to DMB (r2 = 0.33; p = 0.023) and to Young’s modulus (r2 = 0.31; p = 0.032), but not to maximal strength (r2 = 0.14; p = 0.065). Thus, DMB, microhardness and stiffness are correlated at the tissue level. Discussion Parameters reflecting the microhardness of bone and its mineralization were measured in iliac bone samples from control women and men, and women and men with idiopathic osteoporosis. In controls, microhardness was not significantly modified with age. It was not different between cortical and cancellous bone tissues, or between the two cortices. DMB parameters did not change significantly with age except for the Heterogeneity Index that decreased with age (−30% between the first and the fourth quartiles). Such a
homogeneization with an increase of the number of osteons and packet fragments has been reported with age [43]. DMB parameters were not different between cortical and cancellous bone tissues, and between the two cortices. DMB and microhardness values were significantly decreased (−7% and −10%, respectively) in osteoporotic patients compared to controls. When measured separately at the BSU level, there were significant correlations between DMB and microhardness in control and osteoporotic patients. Some measurements have been previously reported on the differences in microhardness in various classes of BSUs chosen according to their qualitative level of mineralization [35,36]. Thus, the level of mineralization is an important determinant of the microhardness of bone tissue, but it does not explain all of its variance. Indeed, microhardness of organic matrix is 3-fold lower than the microhardness of calcified tissue. Finally, we found that bone microhardness is correlated with DMB and apparent Young’s modulus [14]. Bones are known to become more brittle when the mineral content exceeds a critical value and are also less able to bear load when the mineral content is too low [28,44]. The link between mineral content, hardness and elastic modulus has been reported at the tissue level without distinction between the BSUs [28,44]. Variation in mineral content in osteoporosis is important, but there are other mineral and organic matrix determinants that also contribute to the loss of mechanical strength in osteoporotic bone [2]. For example, there is a positive relationship between stiffness and degree of mineralization in woven bone from fetal and neonatal pigs [45]. Quantitative microradiography not only allows the measurement of global but also focal DMB, at the BSU level within the limits imposed by the thickness of the section [7,15,18]. Microscopic mineral variations and mineral density distributions have also been evaluated by quantitative backscattered electron imaging [46,47] and by synchrotron radiation microtomography [10–12], providing values
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similar to those reported here. The DMB of trabecular bone from healthy adult individuals was shown to be constant regardless of gender, age, ethnicity, or skeletal site [46–48]. In adult bone, the major biological determinant of mineralization is the rate of turnover. Thus, an augmentation of the turnover induces a decrease in “lifespan” of BSUs, i.e., of the time available for the secondary mineralization. The new BSUs are therefore eroded before completing their secondary mineralization. The above idea is supported by the presence of a large amount of BSUs that are not completely mineralized and a low mean DMB [49–51]. Conversely, a marked reduction in the “birthrate” of BMUs (antiresorptive agents as bisphosphonates, estrogen, SERMs) prolongs the “lifespan” of the BSUs, allowing a more complete secondary mineralization. This is manifested by an increase in the parameters reflecting the secondary mineralization [6–9,11], except for the heterogeneity index [23]. In one human femur sample, nanohardness has been reported to be smaller in lamellae of osteons than in interstitial bone [52]. Our results confirm that interstitial bone is harder and more mineralized than recently deposited bone tissue. The contribution of the organic matrix (mainly collagen) to material properties of bone has been underappreciated and is still not well studied [53–55]. Our results illustrate that organic matrix accounts for about 1/3 of the hardness of bone tissue. This emphasizes the role of collagen in bone quality. However, we consider that the properties of uncalcified organic matrix are similar to those of the organic part of the calcified matrix, and that these properties are similar between osteomalacia and normal bone. This is probably not the case, but we cannot assess separately the impact of mineralization and of the presence of mineral crystals on the quality of the organic matrix. Finally, the specific role of noncollagenous bone constituents in mineralization has been related [56] and we have to investigate their possible influence on microhardness. There was a close correlation between bone stiffness, yield strength, and ultimate strength for human vertebral cancellous bone in compression that was not statistically different from that for animal cortical bone in tension [57]. Significant correlations between DMB and Young’s modulus and ultimate strength have been reported in cancellous bone from human calcaneus in compression [14]. Finally, in the present study, significant correlations between microhardness and DMB and Young’s modulus are reported in cancellous bone from human calcaneus [14]. Thus, a relationship between microhardness, degree of mineralization, stiffness and strength was clearly supported in human bone samples. This new approach to investigate the determinants of bone quality emphasizes the important role of bone mineral. Beside parameters of DMB, the mechanical properties at tissue level (micro-indentation) have also been assessed. To refine investigations into the role of mineralization as a determinant of bone strength, Fourier transform infrared microspectroscopy (FTIRM) could be used to determine the physicochemical properties of bone constituents. This would allow evaluation of matrix parameters with greater sensitivity in order to characterize the quality of both mineral and collagen in human bone [20]. To conclude, in human bone samples from controls and osteoporotic patients, microhardness and mineralization appear highly but not exclusively correlated. Indeed, the specific influence of organic matrix on microhardness has been illustrated for the first time by our study. Acknowledgments This work was supported by INSERM and in part by an unrestricted educational grant from Eli Lilly to INSERM. The expert technical assistance of Catherine Simi (INSERM Unité 831, Université de Lyon, Faculté de Médecine R. Laennec, Lyon, France) was appreciated. The authors also thank Prof. Mark Forwood (Lyon and Brisbane) for helpful discussions and remarks during the preparation of the manuscript.
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Bone j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b o n e
The role of mineralization and organic matrix in the microhardness of bone tissue from controls and osteoporotic patients G. Boivin a,⁎, Y. Bala a, A. Doublier a, D. Farlay a, L.G. Ste-Marie b, P.J. Meunier a, P.D. Delmas a a b
INSERM Unité 831, Université de Lyon, Faculté de Médecine R. Laennec, Lyon, France Centre de Recherche du CHUM, Hôpital Saint-Luc, Université de Montréal, Montréal, Canada
a r t i c l e
i n f o
Article history: Received 28 January 2008 Revised 27 May 2008 Accepted 28 May 2008 Available online 10 June 2008 Edited by: Rene Rizzoli Keywords: Microhardness Bone mineralization Osteoporosis Vickers indentation Quantitative microradiography
a b s t r a c t Degree of mineralization of bone (DMB) is a major intrinsic determinant of bone strength at the tissue level but its contribution to the microhardness (Vickers indentation) at the intermediary level of organization of bone tissue, i.e., Bone Structural Units (BSUs), has never been assessed. The purpose of this study was to analyze the relationship between the microhardness, the DMB and the organic matrix, measured in BSUs from human iliac bone biopsies. Iliac bone samples from controls and osteoporotic patients (men and women), embedded in methyl methacrylate, were used. Using a Vickers indenter, microhardness (kg/mm2) was measured, either globally on surfaced blocks or focally on 100 μm-thick sections from bone samples (load of 25 g applied during 10 sec; CV = 5%). The Vickers indenter was more suited than the Knoop indenter for a tissue like bone in which components are diversely oriented. Quantitative microradiography performed on 100 μm-thick sections, allowed measurement of parameters reflecting the DMB (g/cm3). Assessed on the whole bone sample, both microhardness and DMB were significantly lower (−10% and −7%, respectively) in osteoporotic patients versus controls (p b 0.001). When measured separately at the BSU level, there were significant positive correlations between microhardness and DMB in controls (r2 = 0.36, p b 0.0001) and osteoporotic patients (r2 = 0.43, p b 0.0001). Mineralization is an important determinant of the microhardness, but did not explain all of its variance. To highlight the role of the organic matrix in bone quality, microhardness of both osteoid and adjacent calcified matrix were measured in iliac samples from subjects with osteomalacia. Microhardness of organic matrix is 3-fold lower than the microhardness of calcified tissue. In human calcanei, microhardness was significantly correlated with DMB (r2 = 0.33, p = 0.02) and apparent Young’s modulus (r2 = 0.26, p = 0.03). In conclusion, bone microhardness measured by Vickers indentation is an interesting methodology for the evaluation of bone strength and its determinants at the BSU level. Bone microhardness is linked to Young’s modulus of bone and is strongly correlated to mineralization, but the organic matrix accounts for about one third of its variance. © 2008 Elsevier Inc. All rights reserved.
Introduction Bone strength is influenced by a number of different determinants, in addition to mass, size, geometry and microarchitecture, bone strength is also influenced by the intrinsic material properties of the tissue. These include the mineralization of bone and the characteristics of the organic matrix (orientation and chemical structure of the collagen fibers), the accumulation of microdamage, and indirectly the apoptosis of osteocytes [1–4]. Bone “quality” is profoundly influenced by the rate of bone turnover [5]. The major role of mineralization has been emphasized by the fact that fracture risk and bone mineral density were changed in osteoporotic patients treated with antiresorptive agents, without modifications of bone
⁎ Corresponding author. INSERM Unité 831, Université de Lyon, Faculté de Médecine R. Laennec, 69372 Lyon Cedex 08, France. Fax: +33 4 78 77 86 63. E-mail address: [email protected] (G. Boivin). 8756-3282/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.05.024
matrix volume or bone microarchitecture. This was largely, if not totally, due to modifications of the duration of secondary mineralization [6–13]. The degree of mineralization of bone (DMB) not only influences the mechanical resistance of bone [14] but also partly determines the bone mineral density [6]. From microradiographic observations [15– 17], it is clear that DMB varies over bone structural units (BSUs, namely the osteons in cortical bone and the trabecular packets in cancellous bone), with the recently deposited ones being much less mineralized than the older ones (Fig. 1). This heterogeneity in the DMB is explained by the fact that bone formation, which follows bone resorption in the remodeling sequence is a multi-step process. Following its deposition, the new matrix begins to mineralize after about 5 to 10 days from the time of deposition, and the linear rate of this primary mineralization can be measured directly in vivo using double tetracycline labeling. After full completion of the BSUs, a phase of secondary mineralization begins [16–18]. This process consists of a slow and gradual maturation of the mineral component, including an
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Fig. 1. Human bone samples. Microradiograph of a 100 ± 1 μm-thick section illustrating the heterogeneity of the mineralization in the various BSUs (young bone) and in the interstitial old bone tissue (left). The brighter interstitial lamellae had a mineral content of about 1.40 g/cm3 and a hardness of about 65 kg/mm2 while the least highly mineralized BSUs had a mineral content of about 1.00 g/cm3 and a hardness of less than 40 kg/mm2. Unstained sections of cortical (upper right) and trabecular (lower right) with the Vickers impressions into bone tissue.
increase in the amount of crystals, an augmentation of crystal size toward their maximum dimensions and an increase in the perfection of the crystals [19,20]. If remodeling activity is not accelerated, this secondary mineralization progressively augments the mineral content of bone matrix, until the maximum mineral content is reached. Unknown in humans, the duration of the secondary mineralization has been reported in rabbits [21] and is now described in an animal model (ewe) having a Haversian remodeling similar to humans [22]. Quantitative microradiography giving a high linear resolution, allowed measurement of the focal DMB of each BSU within the limits imposed by the thickness of the section [15,18,23]. Variations of mineral density have also been evaluated in cancellous bone by others through quantitative backscattered electron imaging [16,24] and, in total bone, by synchrotron radiation microtomography [10,12]. Hardness is a measurement used to assess the resistance of a material deformation, that is to say, the ability of a material's surface to resist indentation with or without permanent indentation [25–27]. Microhardness tests allow the investigation of relationships between the structure and the mechanical characteristics of a material at a microstructural scale. In various bones, microhardness, mineral content and Young’s modulus increase with age [28]. The microhardness test is a valuable tool by which our understanding of the mechanics of bone can be improved. The dimensions of an impression that results from forcing an indenter into the surface of a material, with a known and constant load, lead to the calculation of the hardness value. The pyramidal square-based Vickers indenter has been often used for indenting bone (Fig. 1). It has been suggested that the Knoop indenter has an advantage over the Vickers one (see below) for analyzing cortical bone microhardness [29]. In contrast to nanoindentation for assessing parameters at the lamellar level [30], microhardness measured by microindentation appears most suited to test mechanical properties of bone at the intermediary level of organization of bone, i.e., BSUs. The mineral content of bone is correlated with Young's modulus in cortical bone [31], and microhardness values increase with Young's modulus [32–34]. However, no extensive and comparative analysis of microhardness and mineralization of bone tissue has been done in bone samples. Early studies only
reported partial data (microhardness calculated in different kinds of osteons based on the grey-levels in microradiographs) [35,36]. We hypothesized that a strong relationship exists between microhardness and mineralization. The main purposes of the present study were to investigate parameters reflecting microhardness and mineralization at the BSU level in controls and osteoporotic patients (men and women), and to test the potential relationship between these parameters. The role of organic matrix in the microhardness of bone was also analyzed. Materials and methods Subjects and bone samples We obtained: - iliac bone samples taken at necropsy from subjects who died suddenly without apparent bone disease, representing the control group, 6 women (82 ± 7 years) and 13 men (66 ± 4 years), - iliac bone biopsies from 30 men (48 ± 11 years) suffering from untreated idiopathic osteoporosis [13 from France (50 ± 11 years) and 17 from Canada (49 ± 8 years)], osteoporotic patients have been diagnosed on clinical, radiological, biological and histomorphometric criteria, - iliac bone biopsies taken from 22 untreated women with postmenopausal osteoporosis (62 ± 5 years) corresponding to a subset of a large group [8], - calcanei excised post-mortem from 18 subjects (78 ± 8 years) and previously used for measurement of Young's modulus and maximal strength in compression tests [14], - iliac bone biopsies from 13 patients (47 ± 11 years) suffering from untreated osteomalacia diagnosed on clinical, radiological, biological and histomorphometric criteria. Iliac bone samples were fixed in 70% alcohol, dehydrated in absolute alcohol, then embedded in methyl methacrylate without prior decalcification [37]. For quantitative microradiography, thick sections (about 150 μm) were cut from embedded bone samples with
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a precision diamond wire saw (Well, Escil, Chassieu, France), progressively ground to a thickness of 100 μm, and polished with a diamond paste (1 μm). The thickness of the section was measured with an accuracy of 1 μm using a precision micrometer (Compac, Geneva, Switzerland). After ultrasonic cleaning the bone sections were microradiographed [15,16]. For microhardness tests, either the embedded blocks or the 100 μm-thick sections previously used for microradiography, were meticulously surfaced and polished with an alumina suspension (1 μm). If orientation of the blocks was possible before sectioning, the cutting plane perpendicular to the Haversian canals of cortical bone was preferred. Quantitative microradiography Contact microradiography of 100 μm-thick bone sections was performed using a X-ray diffraction unit PW 1830/40 equipped with a diffraction tube PW 2273/20 (Philips, Limeil Brévannes, France) operating at 25 kV and 25 mA. The nickel-filtered copper Kα radiation was used. A Geola high-resolution film (VRP-M green sensitive) was exposed for 20 min (Slavich International Wholesale Office, Vilnius, Lithuania). For quantitative evaluation of the X-ray absorption by the bone section, aluminum step-wedges were exposed on each microradiograph [15]. The DMB was quantified using a combined contact microradiography, microdensitometry computerized method [15]. This procedure used new automatic programs for analyzing grayness levels (MorphoExpert and Mineralization, ExploraNova, La Rochelle, France). A digital camera (actual resolution: 1600 × 1200 pixels or 800 × 600 after binning) captures the microscopic image of the microradiograph. After calibration using the aluminum reference system, the measured regions of bone tissue are automatically selected, then the gray levels are segmented after bone thresholding. The values of the gray levels are then obtained at pixel level (for a magnification ×2.5, the size of the pixel is 2.82 μm). Finally, gray-level values are converted into DMB values with the construction of a calibration curve based on the measurements obtained on the aluminum step-wedge. DMB are finally expressed in g of mineral over cm3 of bone (g/cm3) and are measured separately in cortical, cancellous, and total (cortical + cancellous) bone tissues. For cortical bone, it represents the mean of the DMB measured separately in the thickest and the thinnest cortices. The main parameters, extracted from DMB measurements, were the mean DMB, the mean highest and most frequent DMB (DMB Freq. Max.) and the mean index of heterogeneity of the distribution of DMB expressed as the mean of the widths at half-maximum measured on the individual DMB curves. Measurement of microhardness Microhardness is measured using a Micromet 5104 (Buehler, Lake Bluff, Illinois, USA) equipped with a Vickers indenter which is more suitable than the Knoop indenter [38]. The microhardness tester is linked to a computer with an OmniMet program allowing the calculation of microhardness and then the processing of the various values measured. The sizes of the impressions are adapted to the study of the intermediary level of organization of bone, i.e. the BSU level. Nanoindentation hardness testing is now used frequently with load forces less than 1 g being applied. However, this does require more stringent environmental control of factors such as vibration, airflow, and temperature. Also, the indentations are typically very small, in bone tissue they are at the level of the lamellae which are not a level of investigation for bone remodeling [27,39,40]. Iliac bone samples and sections are adapted in size and shape to the specimen holders used, and a good surface condition is required to enhance the precision of hardness evaluation. Due to the relatively small size of the impressions created during microhardness tests and because the BSU values were very homogeneous, we were able to take the average of 2 to 5 impressions from any BSU to
give the value of microhardness for that BSU (Fig. 1). In human bone tissue, all the measurements of microhardness are constant in a BSU, but the microhardness differs from one BSU to another in agreement with the wide heterogeneity of the secondary mineralization. The formulae used to calculate the microhardness of the tissue are: for Knoop tests, Hk = 13689.9 P/d2 (where Hk is Knoop microhardness expressed in kg/mm2, P is test load expressed in g, d is length of the longer diagonal expressed in mm), for Vickers tests, Hv = 1854.4 P/d2 (where Hv is Vickers microhardness expressed in kg/mm2, P is test load expressed in g, d is the mean length of the two diagonals expressed in mm). In our laboratory, microhardness tests with Vickers indenter are performed at a load of 25 g for 10 s. This choice was made after testing in the same bone samples, loads of 10, 25, 50 g applied for 5, 10 and 15 s. A combination of 25 g for 10 s was finally chosen for the tests at the surface of blocks and of bone sections. Indeed, the depth of the impression is always inferior to 10 μm. Hence, there was no risk that the impression pierced the section, allowing the hardness of the specimen holder to influence the values. Similar values (load) have been recently applied to bone samples [41,42], and the intraand inter-observer coefficients of variation were ≤5%. In control specimens in which the amount of bone available was larger than in a bone biopsy, faces cut perpendicularly or in parallel to Haversian canals were both used for the estimation of the influence of the orientation of the tissue on the microhardness measurements. For this purpose, a comparison of the values obtained in the same BSUs with Vickers and Knoop indenters was performed. In fact, in each BSU tested, hardness values have been measured with Vickers indenter without particular orientation, with Knoop indenter having their longest diagonal oriented first parallel and second perpendicular to the main arrangement of the lamellae (Fig. 2). Vickers microhardness corresponded to the mean microhardness measured on Knoop impressions oriented perpendicularly or in parallel to bone lamellae. Since no single microhardness measurement can precisely reflect the microhardness of bone tissue as a whole, a study was performed to evaluate the optimum number of impressions required to obtain a correct and reproducible value of the microhardness on the surfaced blocks. Up to 120 different measurements were performed for each sample showing that the mean microhardness did not change after 60 measurements. For example, in control subjects, cortical Hv was 48.84 ± 3.25 kg/mm2 for 100 impressions and 48.83 ± 3.27 kg/mm2 for 40 indentations. Thus, global microhardness calculated at the level of the surface of a bone sample is reported as the mean value of 60 individual
Fig. 2. Bone sample from a control woman with both Vickers and Knoop impressions. In 6 control women, mean Vickers microhardness corresponded to the mean values from Knoop impressions oriented perpendicular and parallel to bone lamellae.
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Table 1 Human control and osteoporotic (OP) patients Human subjects
19 Controls 52 OP patients 30 OP men 22 OP women 13 control men 6 control women
Total bone
Cortical bone
Cancellous bone
Hv25 (kg/mm2)
DMB (g/cm3)
HI (g/cm3)
Hv25 (kg/mm2)
DMB (g/cm3)
HI (g/cm3)
Hv25 (kg/mm2)
DMB (g/cm3)
HI (g/cm3)
49.18 ± 1.82 44.10 ± 4.72⁎ 40.52 ± 1.88# 48.97 ± 2.46 48.96 ± 1.85 49.71 ± 2.01
1.10 ± 0.09 1.03 ± 0.07⁎ 1.02 ± 0.06# 1.05 ± 0.08 1.10 ± 0.09 1.10 ± 0.08
0.22 ± 0.07 0.25 ± 0.07 0.23 ± 0.06 0.28 ± 0.07 0.22 ± 0.08 0.21 ± 0.03
49.30 ± 2.16 44.05 ± 4.69⁎ 40.59 ± 2.07# 48.77 ± 2.66 49.02 ± 2.03 49.91 ± 2.51
1.10 ± 0.09 1.01 ± 0.07⁎ 1.00 ± 0.06# 1.03 ± 0.08 1.10 ± 0.09 1.09 ± 0.08
0.21 ± 0.07 0.24 ± 0.06 0.22 ± 0.05 0.25 ± 0.06 0.21 ± 0.08 0.21 ± 0.04
48.92 ± 1.57 44.23 ± 5.07⁎ 40.28 ± 2.15# 49.30 ± 2.54 48.73 ± 1.62 49.33 ± 1.52
1.11 ± 0.08 1.07 ± 0.08⁎ 1.08 ± 0.09# 1.08 ± 0.08 1.11 ± 0.06 1.12 ± 0.10
0.19 ± 0.05 0.23 ± 0.06⁎ 0.21 ± 0.04 0.24 ± 0.07 0.19 ± 0.06 0.20 ± 0.03
Mean values (±SD) for the Vickers microhardness (Hv25), the degree of mineralization of bone (DMB) and the heterogeneity index of the distribution of DMB (HI). *pb0.001 and epb0.003 versus corresponding controls.
measurements (20 for each cortex and 20 in the cancellous bone), taken at random but separated by at least 500 μm. The distance between impression and the boundary of bone is at least 10 μm. Because the BSU is the intermediary level allowing evaluation of the changes due to remodeling activity, focal microhardness was measured on the 100 μm-thick sections used for microradiography, in order to identify the BSUs according to their DMB (Fig. 1). Newly formed bone (recent BSUs with a low DMB on microradiography) and old bone (mainly interstitial with high to medium DMB), were tested both in cortical and cancellous bone tissues. Finally, the correlation between both microhardness and DMB was tested at the BSU level, to assess the role of mineralization in the hardness of the tissue. Statistical analysis Results were expressed as mean ± standard deviation (SD). According to the number of samples in the groups, parametric (unpaired Student’s t test for the comparison of means) and non-parametric (Mann–Whitney’s U test for the comparison of means, Spearman test for the correlations between two parameters) tests were used. Significant results were accepted when p ≤ 0.05. Results Parameters reflecting the mineralization of bone and its microhardness were measured in iliac bone samples from control men and women, and men and women with idiopathic osteoporosis. DMB and microhardness values were significantly lower in osteoporotic patients compared to controls (Table 1). The decreases (−7%) were observed in both in men and women but the decrease was significant only for men (Table 1). DMB and microhardness did not change with age in controls (p N 0.05 r2 = 0.010 and 0.002, respectively) and
Fig. 3. Measured separately in 185 BSUs from osteoporotic women (black dot, y = 33.31x + 14.87) and 80 BSUs from osteoporotic men (open square, y = 32.71x + 14.33), Vickers microhardness was significantly (p b 0.0001) correlated with the degree of mineralization of bone.
untreated osteoporotic patients (p = 0.76, r2 = 0.003, and p = 0.36, r2 = 0.03, respectively). The highest and most frequent DMB was significantly decreased (−10%, p b 0.003) in osteoporotic patients (1.00 ± 0.09 g/cm3) compared to controls (1.10 ± 0.10 g/cm3). Even if a tendency to the augmentation of the heterogeneity of the mineralization is noted (p = 0.064 in total bone, Table 1), the mean heterogeneity index was not significantly modified. All parameters (mineralization and microhardness) analyzed were similar in the two populations (French and Canadian) of osteoporotic men and were given for the global group of osteoporotic men. Microhardness and parameters reflecting the secondary mineralization of bone were not significantly different when measured separately in cortical and cancellous bone tissues (Table 1). No difference was observed in the two cortices, the thickest and the thinnest (data not shown). Microhardness was lower in recent BSUs than in old interstitial tissue as quantified by focal measurements at the BSU level. When measured separately at BSU level, there were significant positive correlations between microhardness and DMB in controls (r2 = 0.36, p b 0.0001), osteoporotic men (r2 = 0.52, p b 0.0001, Fig. 3) and osteoporotic women (r2 = 0.40, p b 0.0001, Fig. 3). If these two groups of osteoporotic patients were analyzed together representing measurement of 265 BSUs, the correlation was also significant (r2 = 0.43, p b 0.0001). Thus, the level of secondary mineralization appears to be the major cause of change in the microhardness of bone tissue, but mineralization explains only 40 to 50% of the variance of the microhardness of bone tissue. However, for a given value of DMB (Fig. 3), a large variation in microhardness was observed. Conversely, for a given value of microhardness, various DMB values were found. This variation was emphasized by the observation of the individual curves in osteoporotic men (Fig. 4) and women showing similar slopes despite different spectra of DMB.
Fig. 4. In 8 osteoporotic men, there are significant correlations between the Vickers microhardness and the degree of mineralization of bone within subjects, but large between subjects variability (from r2 = 0.80 to 0.76, the regressions equations are the following: y = 0.018x + 0.072, y = 0.019x + 0.058, y = 0.014x + 0.304, y = 0.013x + 0.349, y = 0.022x − 0.046, y = 0.014x + 0.366, y = 0.012x + 0.510, y = 0.022x + 0.133). Similar observations (not shown) were obtained in osteoporotic women.
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Fig. 5. Stained section of bone from a patient with osteomalacia showing both calcified and osteoid tissues (right). Vickers microhardness (Hv), separately measured in calcified matrix (upper left) and in osteoid tissue alone (lower left) in patients with osteomalacia (n = 13), illustrates the role of osteoid tissue that accounted for 36.4 ± 2.7% of microhardness of calcified matrix.
To highligtht the role of organic matrix in bone quality, microhardness of both osteoid and adjacent areas of calcified matrix were measured in iliac bone samples from patients with osteomalacia. Microhardness of osteoid tissue is about 3-fold lower than the total microhardness of calcified matrix located in the vicinity of non mineralized matrix (Fig. 5). Furthermore, this observation was reinforced by the fact that in osteoporotic patients, regressions show that, if a value 0 is attributed to DMB, the microhardness values would be 15.66 and 14.33 kg/mm2 in women and men, respectively (Fig. 3) and 14.85 kg/mm2 in the whole group of osteoporotic patients. These values match closely to the mean value of microhardness measured in osteoid tissue in biopsies from patients diagnosed with osteomalacia (Fig. 5). We analyzed the correlations between mineralization, microhardness measured at the tissue level and biomechanical properties previously measured at organ level in contiguous samples taken from human calcanei mainly constituted of cancellous bone. Microhardness was significantly correlated to DMB (r2 = 0.33; p = 0.023) and to Young’s modulus (r2 = 0.31; p = 0.032), but not to maximal strength (r2 = 0.14; p = 0.065). Thus, DMB, microhardness and stiffness are correlated at the tissue level. Discussion Parameters reflecting the microhardness of bone and its mineralization were measured in iliac bone samples from control women and men, and women and men with idiopathic osteoporosis. In controls, microhardness was not significantly modified with age. It was not different between cortical and cancellous bone tissues, or between the two cortices. DMB parameters did not change significantly with age except for the Heterogeneity Index that decreased with age (−30% between the first and the fourth quartiles). Such a
homogeneization with an increase of the number of osteons and packet fragments has been reported with age [43]. DMB parameters were not different between cortical and cancellous bone tissues, and between the two cortices. DMB and microhardness values were significantly decreased (−7% and −10%, respectively) in osteoporotic patients compared to controls. When measured separately at the BSU level, there were significant correlations between DMB and microhardness in control and osteoporotic patients. Some measurements have been previously reported on the differences in microhardness in various classes of BSUs chosen according to their qualitative level of mineralization [35,36]. Thus, the level of mineralization is an important determinant of the microhardness of bone tissue, but it does not explain all of its variance. Indeed, microhardness of organic matrix is 3-fold lower than the microhardness of calcified tissue. Finally, we found that bone microhardness is correlated with DMB and apparent Young’s modulus [14]. Bones are known to become more brittle when the mineral content exceeds a critical value and are also less able to bear load when the mineral content is too low [28,44]. The link between mineral content, hardness and elastic modulus has been reported at the tissue level without distinction between the BSUs [28,44]. Variation in mineral content in osteoporosis is important, but there are other mineral and organic matrix determinants that also contribute to the loss of mechanical strength in osteoporotic bone [2]. For example, there is a positive relationship between stiffness and degree of mineralization in woven bone from fetal and neonatal pigs [45]. Quantitative microradiography not only allows the measurement of global but also focal DMB, at the BSU level within the limits imposed by the thickness of the section [7,15,18]. Microscopic mineral variations and mineral density distributions have also been evaluated by quantitative backscattered electron imaging [46,47] and by synchrotron radiation microtomography [10–12], providing values
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similar to those reported here. The DMB of trabecular bone from healthy adult individuals was shown to be constant regardless of gender, age, ethnicity, or skeletal site [46–48]. In adult bone, the major biological determinant of mineralization is the rate of turnover. Thus, an augmentation of the turnover induces a decrease in “lifespan” of BSUs, i.e., of the time available for the secondary mineralization. The new BSUs are therefore eroded before completing their secondary mineralization. The above idea is supported by the presence of a large amount of BSUs that are not completely mineralized and a low mean DMB [49–51]. Conversely, a marked reduction in the “birthrate” of BMUs (antiresorptive agents as bisphosphonates, estrogen, SERMs) prolongs the “lifespan” of the BSUs, allowing a more complete secondary mineralization. This is manifested by an increase in the parameters reflecting the secondary mineralization [6–9,11], except for the heterogeneity index [23]. In one human femur sample, nanohardness has been reported to be smaller in lamellae of osteons than in interstitial bone [52]. Our results confirm that interstitial bone is harder and more mineralized than recently deposited bone tissue. The contribution of the organic matrix (mainly collagen) to material properties of bone has been underappreciated and is still not well studied [53–55]. Our results illustrate that organic matrix accounts for about 1/3 of the hardness of bone tissue. This emphasizes the role of collagen in bone quality. However, we consider that the properties of uncalcified organic matrix are similar to those of the organic part of the calcified matrix, and that these properties are similar between osteomalacia and normal bone. This is probably not the case, but we cannot assess separately the impact of mineralization and of the presence of mineral crystals on the quality of the organic matrix. Finally, the specific role of noncollagenous bone constituents in mineralization has been related [56] and we have to investigate their possible influence on microhardness. There was a close correlation between bone stiffness, yield strength, and ultimate strength for human vertebral cancellous bone in compression that was not statistically different from that for animal cortical bone in tension [57]. Significant correlations between DMB and Young’s modulus and ultimate strength have been reported in cancellous bone from human calcaneus in compression [14]. Finally, in the present study, significant correlations between microhardness and DMB and Young’s modulus are reported in cancellous bone from human calcaneus [14]. Thus, a relationship between microhardness, degree of mineralization, stiffness and strength was clearly supported in human bone samples. This new approach to investigate the determinants of bone quality emphasizes the important role of bone mineral. Beside parameters of DMB, the mechanical properties at tissue level (micro-indentation) have also been assessed. To refine investigations into the role of mineralization as a determinant of bone strength, Fourier transform infrared microspectroscopy (FTIRM) could be used to determine the physicochemical properties of bone constituents. This would allow evaluation of matrix parameters with greater sensitivity in order to characterize the quality of both mineral and collagen in human bone [20]. To conclude, in human bone samples from controls and osteoporotic patients, microhardness and mineralization appear highly but not exclusively correlated. Indeed, the specific influence of organic matrix on microhardness has been illustrated for the first time by our study. Acknowledgments This work was supported by INSERM and in part by an unrestricted educational grant from Eli Lilly to INSERM. The expert technical assistance of Catherine Simi (INSERM Unité 831, Université de Lyon, Faculté de Médecine R. Laennec, Lyon, France) was appreciated. The authors also thank Prof. Mark Forwood (Lyon and Brisbane) for helpful discussions and remarks during the preparation of the manuscript.
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