- Email: [email protected]
Reduced tissue hardness of trabecular bone is associated with severe osteoarthritis
Journal of Biomechanics 44 (2011) 1593–1598
Contents lists available at ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Reduced tissue hardness of trabecular bone is associated with severe osteoarthritis ¨ hman a,c, Massimiliano Baleani a,n, Marco Viceconti a Enrico Dall’Ara a,b, Caroline O a
Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Bologna, Italy Institute of Lightweight Design and Structural Biomechanics, Vienna University of Technology, Vienna, Austria c Facolta di Ingegneria, Universita di Bologna, Bologna, Italy b
a r t i c l e i n f o
abstract
Article history: Accepted 21 December 2010
This study investigated whether changes in hardness of human trabecular bone are associated with osteoarthritis. Twenty femoral heads extracted from subjects without musculoskeletal diseases (subject age: 49–83 years) and twenty femoral heads extracted from osteoarthritic subjects (subject age: 42–85 years) were tested. Sixty indentations were performed along the main trabecular direction of each sample at a fixed relative distance. Two microstructures were found on the indenting locations: packs of parallel-lamellae (PL) and secondary osteons (SO). A 25 gf load was applied for 15 s and the Vickers Hardness (HV) was assessed. Trabecular tissue extracted from osteoarthritic subjects was found to be about 13% less hard compared to tissue extracted from non-pathologic subjects. However, tissue hardness was not significantly affected by gender or age. The SO was 10% less hard than the PL for both pathologic and non-pathologic tissues. A hardness of 34.1 HV for PL and 30.8 HV for SO was found for the nonpathologic tissue. For osteoarthritic tissue, the hardness was 30.2 HV for PL and 27.1 HV for SO. In the bone tissue extracted from osteoarthritic subjects the occurrence of indenting a SO (28%) was higher than that observed in the non-pathological tissue (15%). Osteoarthritis is associated with reduced tissue hardness and alterations in microstructure of the trabecular bone tissue. Gender does not significantly affect trabecular bone hardness either in nonpathological or osteoarthritic subjects. A similar conclusion can be drawn for age, although a larger donor sample size would be necessary to definitively exclude the existence of a slight effect. & 2011 Published by Elsevier Ltd.
Keywords: Trabecular bone Osteoarthritis Hardness Microstructure
1. Introduction Osteoarthritis (OA) is a common musculoskeletal disorder with considerable morbidity and mortality (Cooper et al., 1991). OA is generally considered to be a disease of the articular cartilage. It has been hypothesised that the healing of microfractures, caused by the overloaded cartilage in the subchondral bone, might increase the bone stiffness in this area (Radin et al., 1972). However, it has been suggested that it might as well be the underlying subchondral bone that plays a significant role in the attenuation of the loads applied to the joint that is first affected by this disease (Fazzalari et al., 1987; Radin et al., 1970). Whichever is the pathogenesis of OA, it has been demonstrated that this disease affects the trabecular bone. In fact, it has been found that both the ratio between mineral and collagen
n
Corresponding author. Tel.: +39 051 6366864; fax: + 39 051 6366863. E-mail address: [email protected] (M. Baleani).
0021-9290/$ - see front matter & 2011 Published by Elsevier Ltd. doi:10.1016/j.jbiomech.2010.12.022
(Brown et al., 2002) and the material density (Li and Aspden, 1997) are lower in OA subjects in comparison with subjects without pathologies. Furthermore, a biochemical analysis of the human femoral head suggests that the trabecular bone matrix in OA patients is subjected to an increased turnover (Mansell et al., 1997) and is metabolically very active (Mansell and Bailey, 1998), compared to non-pathological tissue. The changes in mineralisation and remodelling rate are associated with an alteration of trabecular bone microarchitecture. In particular, OA causes a decrease in trabecular number and an increase in trabecular thickness (Fazzalari and Parkinson, 1998; Perilli et al., 2007), which seem more marked in the weight-bearing region (Neilson et al., 2004). However, it is still not clear whether these effects lead to a similar (Perilli et al., 2007) or a higher trabecular bone apparent density (Li and Aspden, 1997) at a macroscopic scale. At a nanometric scale bone is a composite material made of a stiff inorganic mineral phase of hydroxyapatite together with a softer organic phase (principally collagen type-I) and water (Currey, 2003; Fratzl et al., 2004).
1594
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598
An accepted method to evaluate the mechanical properties of bone at this level is to perform indentation tests (Zysset, 2009). While nanoindentation has been intensively used to assess hardness and Young’s modulus of a single lamella (Hengsberger et al., 2002; Rho et al., 1997; Zysset et al., 1999), hardness at the bone structural unit (i.e. a microstructure of a few lamellae, 50–100 mm—here referred to as bone structural unit, BSU) has been evaluated by micro-indentation. Hardness can be considered as one of the characteristics influencing bone quality (Bouxsein, 2003). Furthermore, it has been shown to correlate with mineral content, elastic modulus, and fracture toughness (Evans et al., 1990; Phelps et al., 2000; Zioupos et al., 2000). Additionally, this property has been used to evaluate the post-surgery effect on the mechanical resistance near the bone-implant interface (Huja et al., 1998; Stea et al., 1995). Surprisingly, little is known about the effect of OA on the mechanical properties of a BSU, which is the basic composite material of trabecular bone tissue. Only one study, known to the authors, has evaluated the effect of OA and osteoporosis on the hardness of human trabecular bone (Coats et al., 2003). However, in that study a control group was not tested. Furthermore, only embedded specimens were considered, whereas it has been demonstrated that embedding significantly affects the hardness of human trabecular bone and it is preferable to test specimens in a wet condition (Dall’Ara et al., 2007). The aim of this study was to evaluate whether changes in the quality of a BSU of trabecular bone tissue, evaluated in terms of hardness, are associated with severe OA. In addition, it was investigated whether the hardness of human trabecular bone is influenced by age and gender.
2. Materials and methods 2.1. Samples Prior to sample collection a written informed consent was obtained from all subjects. A total of 20 human femoral heads were obtained from the International Institute for the Advancement of Medicine (IIAM, Jessup, PA, USA). None of the donors suffered from any bone or cartilage diseases and were therefore considered as a control group (CG). Due to difficulties in collecting bone samples, it was not possible to balance the gender (15 males, 5 females). Furthermore, 20 human femoral heads from osteoarthritic subjects (OAG), undergoing total hip arthroplasty, were chosen to obtain age-matched groups. All samples in the OAG showed altered shape of the femoral head and furthermore a part of the cartilage layer was completely worn down, confirming a severe grade (or grade 4 of the Kellgren and Lawrence scale) of osteoarthritis. For the OAG it was possible to balance the gender (10 males, 10 females). Age details are reported in Table 1. No significant difference in age was found splitting the samples for gender and pathology (ANOVA, p¼ 0.39).
solution for a minimum period of four weeks before testing to reduce the risk of transmission of infectious diseases. It has been previously demonstrated that this treatment has no significant effect on the elastic properties of trabecular bone (Linde and Sorensen, 1993). The procedure used in this study for micro-indentation on wet specimens of human trabecular bone is described in detail in a previous report (Dall’Ara et al., 2007); nevertheless the most important steps are reported. Each femoral head was cut parallel to the frontal plane along the main trabecular direction (Ohman et al., 2007) into two halves. After cutting, one part of each specimen was re-hydrated in Ringer’s solution for at least 24 h to assure a wet condition of the tissue (Dall’Ara et al., 2007). Increasingly fine sandpaper and diamond pastes were used to polish the plane surface revealing the trabeculae. All polishing was performed under constant water irrigation. Between each polishing series, the specimen was cleaned by rinsing it in water and thereafter by means of an air jet. A visual check, using a microscope, was done to assure a good quality of the polished surface.
2.3. Indentation measurements Bone tissue hardness was measured by means of a load controlled Vickers diamond micro-indenter (Leica VMHT). According to the study of Dall’Ara et al. (2007) a load of 25 gf was applied on the indenter for 15 s. Therefore, Vickers Hardness HV/0.025 was measured, although the nomenclature HV (i.e. excluding the indication of the applied force) was preferred to lighten the text. The dimensions of the indentation were measured with an optical microscope after the elastic release of the structure (Fig. 3). The deformation of the indented trabecular, not supported by any embedding material, was estimated (using the theory of elastic beams, with a trabecular length of 1.0 mm, a trabecular diameter of 0.1 mm, an elastic modulus of 15 GPa and an indentation of 50 mm). It was found that the error due to the elastic return was much smaller than the uncertainty deriving from the measurements of the diagonals, which was about 1%. During indentation series the specimens were never left in air for more than 45 min to ensure that they did not completely dehydrate again (Dall’Ara et al., 2007; Johnson and Rapoff, 2007). A total of 60 indentations were performed on the plane surface of each sample on two parallel straight lines, at a distance of 10 mm from each other, along the main trabecular direction (Fig. 1). The first indentation was located approximately 5 mm below the joint surface. A total of 30 indentations were performed on each line at a relative distance of 0.87 0.2 mm from each other (depending on the presence of tissue in the target location) to cover the whole length of the femoral head. This scheme was applied: (1) to avoid the influence of the indentation site on hardness, as it has been demonstrated that hardness changes in different regions of the femoral head (Coats et al., 2003); (2) to assure a minimum distance of 3 diagonals (i.e. 120 mm since an indentation diagonal was about 40 mm) between indentations (ASTM E384). The final location of the indentation was
2.2. Specimen preparation The femoral heads obtained from total hip arthroplasty patients were immersed in 70% ethanol solution after removal (within 12 h) and kept immersed until the beginning of the sample preparation. The femoral heads obtained from IIAM were frozen at 20 1C directly after harvesting, performed within 24–96 h post-mortem (NB within 12 hours post-mortem the corpse was put in a refrigerator at 4 1C) and kept frozen during shipping. Thereafter, the samples were thawed and immersed in 70% ethanol solution. All samples were kept in ethanol Table 1 Mean age, with standard deviation and range, of the subjects included in the four groups. Group
Gender
N
Age
Age min
Age max
CG
F M F M
5 15 10 10
64 7 14 69 7 10 61 7 11 65 7 13
49 51 42 47
83 82 75 85
OAG
Fig. 1. Scheme showing the locations of the 60 indentations performed on each femoral head.
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598 defined with respect to a minimum distances between indentation-tissue edge/ pore of 2.5 diagonals (100 mm) (Johnson and Rapoff, 2007). On the polished surface of the trabecular structure, two different microstructures were found: packs of parallel-lamellae (PL, Fig. 2) and secondary osteons (SO, Fig. 2) (Lozupone and Favia, 1990; Sato et al., 1986). SO and trabecular cross sections (which are similar in appearance) were distinguished by the presence of a vessel channel at the centre of the SO. Considering the fact that indentations were performed along the main trabecular direction and perpendicular to a frontal plane, most trabeculae were cut roughly longitudinally, and trabecular cross sections were not
1595
investigated in the present study. Indentations were done on the target location despite the type of microstructure, which was registered together with the hardness value. In four cases, in which the femoral heads were too small, it was not possible to perform all the 60 indentations. Therefore, these specimens were indented only 50 times. A total of 2360 indentations were done. All indentations where one diagonal was 4 15% longer than the other were discharged, as suggested by Hodgskinson et al. (1989), leaving 2220 valid measurements.
2.4. Statistical analysis The Chauvenet criterion was applied once to all valid measurements to exclude outliers. Since numerous hardness measurements were performed on the same sample, a repeated measures ANOVA was used. Within-sample effect (i.e. indentation location or distance from joint surface) and among-samples effects (i.e. pathology, gender, and age) on hardness were investigated. Furthermore, the possible interactions among these variables were analysed. To allow the analysis, the age values were divided into three groups with 15 years intervals (41–55; 56–70; 71–85) (Wolfram et al., 2009). A w2-test was done to evaluate whether there were differences in distribution of the two structures between the groups. A multifactorial ANOVA was used to evaluate differences in hardness between the two types of structure, only considering the significant parameters highlighted in the repeated measures ANOVA.
3. Results A total of 35 hardness values, out of 2220, were excluded for the Chauvenet criterion. Average hardness values versus age and the respective regression line for the two groups, split by gender, were plotted in Fig. 4. No within-sample effect (i.e. distance from joint surface) or any interactions with the investigated parameters were found (ANOVA: p values in the range 0.34–0.99). It was found that the hardness was not dependant on gender or age (ANOVA: p¼0.98 and p¼0.13, respectively). Conversely, the hardness of the CG (33.575.9 HV) was about 13% higher (ANOVA: p o0.001) compared to the hardness of the OAG (29.376.2 HV). No interactions among investigated parameters were found (p values in the range of 0.28–0.86). A difference in distribution of the two microstructures was found between CG and OAG (w2-test: p o0.001). In fact, SO was observed in 15% of the observations in CG versus 28% in OAG. The data, grouped according to pathology and structure are reported in Fig. 5. A difference in hardness was found between the microstructures (ANOVA po0.001). In particular the PL were about 10% harder than the SO for both pathological and
Fig. 2. Examples of both the structures found during the tests. In particular both a secondary osteon (SO) and a microstructure composed by parallel-lamellae (PL) are indicated.
Fig. 3. Example of an indentation being measured on the trabecular bone tissue surface.
1596
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598
Fig. 4. Hardness values versus age grouped according to pathology and gender.
Fig. 5. Hardness values of the groups split by pathology and microstructure. Bars represent standard deviations. The sample size is indicated in each block.
non-pathological tissues. Moreover, the hardness of both microstructures of the CG (34.0 75.7 HV for PL and 30.976.6 HV for SO) was about 13% higher compared to the hardness of the OAG (30.2 75.7 HV for PL and 27.1 76.9 HV for SO). 4. Discussion The aim of this study was to evaluate whether reduced bone tissue quality, evaluated in term of hardness, is associated with severe osteoarthritis. Furthermore, it was also investigated whether age and gender of the subjects affect the hardness of trabecular tissue. In this study 20 femoral heads extracted from subjects without any bone or cartilage diseases and 20 femoral heads extracted from osteoarthritic subjects were tested by means of microindentation. In the range considered in this study (from 42 to 85 years), age was not correlated with hardness. It should be highlighted that only 40 subjects were included in this study due to difficulties in collecting bone samples, especially from non-pathological subjects. Additionally, only four specimens out of 40 were collected from subjects more than 80 years old (80, 82, 83, and 85), among
which only one female subject was included. Therefore, a slight potential decline in trabecular tissue hardness cannot be definitively excluded. However, the absence of a correlation between hardness and age (in the range 40–80) is in agreement with the study of Weaver (1966) in which it was found that, after bone maturation (around 25–30 years), the mineralisation of the interstitial tissue reaches an equilibrium and the hardness remains constant. Moreover, it has been found that the tissue elastic modulus of trabecular bone is correlated to its hardness (Hoffler et al., 2005). Considering this fact, the result of the present study is in line with the findings of Wolfram et al. (2009), which found no correlation between age and tissue elastic modulus measured in human vertebral trabecular bone tissue. No difference in hardness was found between the genders. This result is in agreement with previous studies in which indentation tests were performed on human bone tissue (Coats, et al., 2003; Hoffler et al., 2000a, 2000b; Weaver, 1966; Wolfram, et al., 2009). Both results are in line with the study of Perilli et al. (2007), in which it was shown that mechanical strength of trabecular human bone, extracted from osteoarthritic subjects, is not significantly affected by age or gender. Conversely, subjects with severe OA were found to have a reduced hardness of about 13% in the trabecular bone tissue of the femoral head, both pooling and splitting the two structures PL and SO. Hence, the different distributions of PL and SO found in the two groups (OAG and CG) did not bias the difference in hardness. This reduction in mechanical properties at the microstructural level is in line with the study of Brown et al. (2002), which found an altered matrix composition and a reduced compressive modulus in samples extracted from osteoarthritic subjects compared to normal ones with similar apparent density. Moreover, Li and Aspden (1997) found a change in the bone composition of osteoarthritic subjects and in particular an increase in water content and a decrease in mineral content compared to subjects without any cartilage or bone diseases. Higher water content and lower mineralisation might lead to a less hard tissue. Additionally, the PL had higher hardness ( + 10%) than the SO both in non-pathologic and pathologic tissue. This result might be explained by the spatial organisation of the lamellae. Furthermore, it was observed that in tissue extracted from subjects with OA the probability of indenting a SO was higher than a PL. It has been suggested that an increment of bone remodelling is seen in osteoarthritic subjects (Bailey et al., 2004; Bettica et al., 2002; Lavigne et al., 2005). Consequently, hypothetically osteoarthritic bone tissue might be composed of a larger number of younger, less mineralised and less hard structures. Therefore, the difference found in hardness of the SO and PL might also be partially due to a different degree of mineralisation of the two microstructures. However, this hypothesis should be verified with a detailed analysis of mineral content in both types of structures. The distance from the joint surface did not affect the hardness in none of the groups. This was unexpected in the OAG, considering that OA involves degeneration of the tissues of the hip joint. However, in this study only severe OA was investigated. Studying different grades of OA would be necessary to understand how the distance from the joint surface affects the bone hardness and could supply more details about the OA pathogenesis. In fact, whether all the above-mentioned changes are causes or consequences of OA is not clear, yet. There is only one study known to the authors that has compared the hardness of human trabecular bone extracted from subjects with different pathologies (Coats et al., 2003). In that study, trabecular tissues extracted from subjects affected by OA and subjects affected by osteoporosis were tested. For the osteoarthritic subjects an average of 42.275.8 HV was found, which is about 45% higher than the average value in the present
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598
study. This disagreement might be explained by the different preparations of the tested specimens. In the study of Coats et al. (2003) the tissue was embedded in acrylic resin before testing whereas in this study the specimens were tested hydrated without previous embedding, as recommended by Dall’Ara et al. (2007). The different procedures may explain a difference of about 30–40% (Dall’Ara et al., 2007; Weaver, 1966). The residual difference might be due to the different applied loads and/or the locations selected for the indentations. In that study in fact a smaller applied load was used and indentations were performed only in two small and localised areas of the femoral head, while in this study the whole head was investigated along the main trabecular direction. An indirect comparison for the CG can be obtained confronting the present results with those obtained by nanoindentation. The hardness found in the CG (33.5 HV equivalent to 0.329 GPa) seems to be in line with the hardness values of femoral neck trabecular bone, derived from the graphs reported by Zysset et al. (1999) and Hoffler et al. (2000a, 2000b). The small differences found might be due to the different anatomical sites indented and the different protocols used. The present study has four main limitations: the low number of non-pathological female subjects from whom specimens were retrieved, the unavailability of specimens from old subjects ( 480 years old), the absence of indentations on trabecular cross sections, and the absence of trabecular tissue samples collected from subject with OA at different stages. Despite these limitations the collected data showed an effect of OA on the trabecular bone of the femoral head, whereas a definitive conclusion about the effect of age on hardness would require a larger donor sample size. Additionally, the availability of tissue sample collected at different degrees of OA would allow investigating the OA pathogenesis, i.e. whether the reduced quality of the tissue is a cause or a consequence of the pathology. In conclusion considering trabecular bone tissue from the femoral head it was found that:
The tissue hardness of osteoarthritic subjects is about 13% lower than that of normal subjects.
In osteoarthritic subjects the probability to find a circular
lamellae microstructure is higher than in non-pathologic subjects. Parallel-lamellae microstructure is harder (+ 10%) than circular-lamellae microstructure for both osteoarthritic and nonpathological subjects. Hardness is not dependent on gender. A larger donor sample size would be necessary to definitively exclude the existence of an effect of age.
Conflict of interest statement None declared.
Acknowledgments This work was partially supported by the European Community (project number: IST-2006-035763; project title: 3D Anatomical Human and project number; IST-2004-026932; project title: Living Human Digital Library; acronym: LHDL). The authors wish to thank Barbara Bordini for the statistical analysis and Luigi Lena for the illustrations.
1597
References Bailey, A.J., Mansell, J.P., Sims, T.J., Banse, X., 2004. Biochemical and mechanical properties of subchondral bone in osteoarthritis. Biorheology 41, 349–358. Bettica, P., Cline, G., Hart, D.J., Meyer, J., Spector, T.D., 2002. Evidence for increased bone resorption in patients with progressive knee osteoarthritis: longitudinal results from the Chingford study. Arthritis and Rheumatism 46, 3178–3184. Bouxsein, M.L., 2003. Bone quality: where do we go from here? Osteoporosis International 14, S118–S127. Brown, S.J., Pollintine, P., Powell, D.E., Davie, M.W., Sharp, C.A., 2002. Regional differences in mechanical and material properties of femoral head cancellous bone in health and osteoarthritis. Calcified Tissue International 71, 227–234. Coats, A.M., Zioupos, P., Aspden, R.M., 2003. Material properties of subchondral bone from patients with osteoporosis or osteoarthritis by microindentation testing and electron probe microanalysis. Calcified Tissue International 73, 66–71. Cooper, C., Cook, P.L., Osmond, C., Fisher, L., Cawley, M.I., 1991. Osteoarthritis of the hip and osteoporosis of the proximal femur. Annals of the Rheumatic Diseases 50, 540–542. Currey, J.D., 2003. Role of collagen and other organics in the mechanical properties of bone. Osteoporosis International 14 (Suppl. 5), 29–36. Dall’Ara, E., Ohman, C., Baleani, M., Viceconti, M., 2007. The effect of tissue condition and applied load on Vickers hardness of human trabecular bone. Journal of Biomechanics 40, 3267–3270. Evans, F.G., Behiri, J.C., Currey, J.D., Bonfield, W., 1990. Microhardness and Young’s modulus in cortical bone exhibiting a wide range of mineral volume fractions, and in a bone analogue. Journal of Materials Science: Materials in Medicine 1, 38–43. Fazzalari, N.L., Parkinson, I.H., 1998. Femoral trabecular bone of osteoarthritic and normal subjects in an age and sex matched group. Osteoarthritis and Cartilage 6, 377–382. Fazzalari, N.L., Vernon-Roberts, B., Darracott, J., 1987. Osteoarthritis of the hip. Possible protective and causative roles of trabecular microfractures in the head of the femur. Clinical Orthopaedics and Related Research 216, 224–233. Fratzl, P., Gupta, H.S., Paschalis, E.P., Roschger, P., 2004. Structure and mechanical quality of the collagen–mineral nano-composite in bone. Journal of Materials Chemistry 14, 2115–2123. Hengsberger, S., Kulik, A., Zysset, P., 2002. Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. Bone 30, 178–184. Hodgskinson, R., Currey, J.D., Evans, G.P., 1989. Hardness, an indicator of the mechanical competence of cancellous bone. Journal of Orthopaedic Research 7, 754–758. Hoffler, C.E., Guo, X.E., Zysset, P.K., Goldstein, S.A., 2005. An application of nanoindentation technique to measure bone tissue Lamellae properties. Journal of Biomechanical Engineering 127, 1046–1053. Hoffler, C.E., Moore, K.E., Kozloff, K., Zysset, P.K., Goldstein, S.A., 2000a. Age, gender, and bone lamellae elastic moduli. Journal of Orthopaedic Research 18, 432–437. Hoffler, C.E., Moore, K.E., Kozloff, K., Zysset, P.K., Brown, M.B., Goldstein, S.A., 2000b. Heterogeneity of bone lamellar-level elastic moduli. Bone 26, 603–609. Huja, S.S., Katona, T.R., Moore, B.K., Roberts, W.E., 1998. Microhardness and anisotropy of the vital osseous interface and endosseous implant supporting bone. Journal of Orthopaedic Research 16, 54–60. Johnson, W.M., Rapoff, A.J., 2007. Microindentation in bone: Hardness variation with five independent variables. Journal of Materials Science: Materials in Medicine 18, 591–597. Lavigne, P., Benderdour, M., Lajeunesse, D., Reboul, P., Shi, Q., Pelletier, J.P., MartelPelletier, J., Fernandes, J.C., 2005. Subchondral and trabecular bone metabolism regulation in canine experimental knee osteoarthritis. Osteoarthritis Cartilage 13, 310–317. Li, B., Aspden, R.M., 1997. Composition and mechanical properties of cancellous bone from the femoral head of patients with osteoporosis or osteoarthritis. Journal of Bone and Mineral Reasearch 12, 641–651. Linde, F., Sorensen, H.C., 1993. The effect of different storage methods on the mechanical properties of trabecular bone. Journal of Biomechanics 26, 1249–1252. Lozupone, E., Favia, A., 1990. The structure of the trabeculae of cancellous bone. 2. Long bones and mastoid. Calcified Tissue International 46, 367–372. Mansell, J.P., Bailey, A.J., 1998. Abnormal cancellous bone collagen metabolism in osteoarthritis. Journal of Clinical Investigation 101, 1596–1603. Mansell, J.P., Tarlton, J.F., Bailey, A.J., 1997. Biochemical evidence for altered subchondral bone collagen metabolism in osteoarthritis of the hip. British Journal of Rheumatology 36, 16–19. Neilson, M., White, A., Malik, U., Morrison, E., McGill, P.E., McDonald, S.W., 2004. Changes in bone architecture in the femoral head and neck in osteoarthritis. Clinical Anatomy 17, 378–391. Ohman, C., Baleani, M., Perilli, E., Dall’Ara, E., Tassani, S., Baruffaldi, F., Viceconti, M., 2007. Mechanical testing of cancellous bone from the femoral head: experimental errors due to off-axis measurements. Journal of Biomechanics 40, 2426–2433. Perilli, E., Baleani, M., Ohman, C., Baruffaldi, F., Viceconti, M., 2007. Structural parameters and mechanical strength of cancellous bone in the femoral head in osteoarthritis do not depend on age. Bone 41, 760–768. Phelps, J.B., Hubbard, G.B., Wang, X., Agrawal, C.M., 2000. Microstructural heterogeneity and the fracture toughness of bone. Journal of Biomedical Materials Research 51, 735–741.
1598
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598
Radin, E.L., Paul, I.L., Tolkoff, M.J., 1970. Subchondral bone changes in patients with early degenerative joint disease. Arthritis and Rheumatism 13, 400–405. Radin, E.R., Paul, I.L., Rose, R.M., 1972. Pathogenesis of primary osteoarthritis. Lancet 1, 1395–1396. Rho, J.Y., Tsui, T.Y., Pharr, G.M., 1997. Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation. Biomaterials 18, 1325–1330. Sato, K., Wakamatsu, E., Sato, T., Honma, T., Kotake, H., Byers, P.D., 1986. Histomorphometric study of trabecular channels in normal iliac bone. Calcified Tissue International 39, 2–7. Stea, S., Visentin, M., Savarino, L., Ciapetti, G., Donati, M.E., Moroni, A., Caja, V., Pizzoferrato, A., 1995. Microhardness of bone at the interface with ceramic-coated metal implants. Journal of Biomedical Materials Research 29, 695–699.
Weaver, J.K., 1966. The microscopic hardness of bone. Journal of Bone and Joint Surgery. American Volume 48, 273–288. Wolfram, U., Wilke, H.J., Zysset, P.K., 2009. Rehydration of vertebral trabecular bone: influences on its anisotropy, its stiffness and the indentation work with a view to age, gender and vertebral level. Bone 46, 348–354. Zioupos, P., Currey, J.D., Casinos, A., 2000. Exploring the effects of hypermineralisation in bone tissue by using an extreme biological example. Connective Tissue Research 41, 229–248. Zysset, P.K., 2009. Indentation of bone tissue: a short review. Osteoporosis International 20, 1049–1055. Zysset, P.K., Guo, X.E., Hoffler, C.E., Moore, K.E., Goldstein, S.A., 1999. Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur. Journal of Biomechanics 32, 1005–1012.
Contents lists available at ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Reduced tissue hardness of trabecular bone is associated with severe osteoarthritis ¨ hman a,c, Massimiliano Baleani a,n, Marco Viceconti a Enrico Dall’Ara a,b, Caroline O a
Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Bologna, Italy Institute of Lightweight Design and Structural Biomechanics, Vienna University of Technology, Vienna, Austria c Facolta di Ingegneria, Universita di Bologna, Bologna, Italy b
a r t i c l e i n f o
abstract
Article history: Accepted 21 December 2010
This study investigated whether changes in hardness of human trabecular bone are associated with osteoarthritis. Twenty femoral heads extracted from subjects without musculoskeletal diseases (subject age: 49–83 years) and twenty femoral heads extracted from osteoarthritic subjects (subject age: 42–85 years) were tested. Sixty indentations were performed along the main trabecular direction of each sample at a fixed relative distance. Two microstructures were found on the indenting locations: packs of parallel-lamellae (PL) and secondary osteons (SO). A 25 gf load was applied for 15 s and the Vickers Hardness (HV) was assessed. Trabecular tissue extracted from osteoarthritic subjects was found to be about 13% less hard compared to tissue extracted from non-pathologic subjects. However, tissue hardness was not significantly affected by gender or age. The SO was 10% less hard than the PL for both pathologic and non-pathologic tissues. A hardness of 34.1 HV for PL and 30.8 HV for SO was found for the nonpathologic tissue. For osteoarthritic tissue, the hardness was 30.2 HV for PL and 27.1 HV for SO. In the bone tissue extracted from osteoarthritic subjects the occurrence of indenting a SO (28%) was higher than that observed in the non-pathological tissue (15%). Osteoarthritis is associated with reduced tissue hardness and alterations in microstructure of the trabecular bone tissue. Gender does not significantly affect trabecular bone hardness either in nonpathological or osteoarthritic subjects. A similar conclusion can be drawn for age, although a larger donor sample size would be necessary to definitively exclude the existence of a slight effect. & 2011 Published by Elsevier Ltd.
Keywords: Trabecular bone Osteoarthritis Hardness Microstructure
1. Introduction Osteoarthritis (OA) is a common musculoskeletal disorder with considerable morbidity and mortality (Cooper et al., 1991). OA is generally considered to be a disease of the articular cartilage. It has been hypothesised that the healing of microfractures, caused by the overloaded cartilage in the subchondral bone, might increase the bone stiffness in this area (Radin et al., 1972). However, it has been suggested that it might as well be the underlying subchondral bone that plays a significant role in the attenuation of the loads applied to the joint that is first affected by this disease (Fazzalari et al., 1987; Radin et al., 1970). Whichever is the pathogenesis of OA, it has been demonstrated that this disease affects the trabecular bone. In fact, it has been found that both the ratio between mineral and collagen
n
Corresponding author. Tel.: +39 051 6366864; fax: + 39 051 6366863. E-mail address: [email protected] (M. Baleani).
0021-9290/$ - see front matter & 2011 Published by Elsevier Ltd. doi:10.1016/j.jbiomech.2010.12.022
(Brown et al., 2002) and the material density (Li and Aspden, 1997) are lower in OA subjects in comparison with subjects without pathologies. Furthermore, a biochemical analysis of the human femoral head suggests that the trabecular bone matrix in OA patients is subjected to an increased turnover (Mansell et al., 1997) and is metabolically very active (Mansell and Bailey, 1998), compared to non-pathological tissue. The changes in mineralisation and remodelling rate are associated with an alteration of trabecular bone microarchitecture. In particular, OA causes a decrease in trabecular number and an increase in trabecular thickness (Fazzalari and Parkinson, 1998; Perilli et al., 2007), which seem more marked in the weight-bearing region (Neilson et al., 2004). However, it is still not clear whether these effects lead to a similar (Perilli et al., 2007) or a higher trabecular bone apparent density (Li and Aspden, 1997) at a macroscopic scale. At a nanometric scale bone is a composite material made of a stiff inorganic mineral phase of hydroxyapatite together with a softer organic phase (principally collagen type-I) and water (Currey, 2003; Fratzl et al., 2004).
1594
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598
An accepted method to evaluate the mechanical properties of bone at this level is to perform indentation tests (Zysset, 2009). While nanoindentation has been intensively used to assess hardness and Young’s modulus of a single lamella (Hengsberger et al., 2002; Rho et al., 1997; Zysset et al., 1999), hardness at the bone structural unit (i.e. a microstructure of a few lamellae, 50–100 mm—here referred to as bone structural unit, BSU) has been evaluated by micro-indentation. Hardness can be considered as one of the characteristics influencing bone quality (Bouxsein, 2003). Furthermore, it has been shown to correlate with mineral content, elastic modulus, and fracture toughness (Evans et al., 1990; Phelps et al., 2000; Zioupos et al., 2000). Additionally, this property has been used to evaluate the post-surgery effect on the mechanical resistance near the bone-implant interface (Huja et al., 1998; Stea et al., 1995). Surprisingly, little is known about the effect of OA on the mechanical properties of a BSU, which is the basic composite material of trabecular bone tissue. Only one study, known to the authors, has evaluated the effect of OA and osteoporosis on the hardness of human trabecular bone (Coats et al., 2003). However, in that study a control group was not tested. Furthermore, only embedded specimens were considered, whereas it has been demonstrated that embedding significantly affects the hardness of human trabecular bone and it is preferable to test specimens in a wet condition (Dall’Ara et al., 2007). The aim of this study was to evaluate whether changes in the quality of a BSU of trabecular bone tissue, evaluated in terms of hardness, are associated with severe OA. In addition, it was investigated whether the hardness of human trabecular bone is influenced by age and gender.
2. Materials and methods 2.1. Samples Prior to sample collection a written informed consent was obtained from all subjects. A total of 20 human femoral heads were obtained from the International Institute for the Advancement of Medicine (IIAM, Jessup, PA, USA). None of the donors suffered from any bone or cartilage diseases and were therefore considered as a control group (CG). Due to difficulties in collecting bone samples, it was not possible to balance the gender (15 males, 5 females). Furthermore, 20 human femoral heads from osteoarthritic subjects (OAG), undergoing total hip arthroplasty, were chosen to obtain age-matched groups. All samples in the OAG showed altered shape of the femoral head and furthermore a part of the cartilage layer was completely worn down, confirming a severe grade (or grade 4 of the Kellgren and Lawrence scale) of osteoarthritis. For the OAG it was possible to balance the gender (10 males, 10 females). Age details are reported in Table 1. No significant difference in age was found splitting the samples for gender and pathology (ANOVA, p¼ 0.39).
solution for a minimum period of four weeks before testing to reduce the risk of transmission of infectious diseases. It has been previously demonstrated that this treatment has no significant effect on the elastic properties of trabecular bone (Linde and Sorensen, 1993). The procedure used in this study for micro-indentation on wet specimens of human trabecular bone is described in detail in a previous report (Dall’Ara et al., 2007); nevertheless the most important steps are reported. Each femoral head was cut parallel to the frontal plane along the main trabecular direction (Ohman et al., 2007) into two halves. After cutting, one part of each specimen was re-hydrated in Ringer’s solution for at least 24 h to assure a wet condition of the tissue (Dall’Ara et al., 2007). Increasingly fine sandpaper and diamond pastes were used to polish the plane surface revealing the trabeculae. All polishing was performed under constant water irrigation. Between each polishing series, the specimen was cleaned by rinsing it in water and thereafter by means of an air jet. A visual check, using a microscope, was done to assure a good quality of the polished surface.
2.3. Indentation measurements Bone tissue hardness was measured by means of a load controlled Vickers diamond micro-indenter (Leica VMHT). According to the study of Dall’Ara et al. (2007) a load of 25 gf was applied on the indenter for 15 s. Therefore, Vickers Hardness HV/0.025 was measured, although the nomenclature HV (i.e. excluding the indication of the applied force) was preferred to lighten the text. The dimensions of the indentation were measured with an optical microscope after the elastic release of the structure (Fig. 3). The deformation of the indented trabecular, not supported by any embedding material, was estimated (using the theory of elastic beams, with a trabecular length of 1.0 mm, a trabecular diameter of 0.1 mm, an elastic modulus of 15 GPa and an indentation of 50 mm). It was found that the error due to the elastic return was much smaller than the uncertainty deriving from the measurements of the diagonals, which was about 1%. During indentation series the specimens were never left in air for more than 45 min to ensure that they did not completely dehydrate again (Dall’Ara et al., 2007; Johnson and Rapoff, 2007). A total of 60 indentations were performed on the plane surface of each sample on two parallel straight lines, at a distance of 10 mm from each other, along the main trabecular direction (Fig. 1). The first indentation was located approximately 5 mm below the joint surface. A total of 30 indentations were performed on each line at a relative distance of 0.87 0.2 mm from each other (depending on the presence of tissue in the target location) to cover the whole length of the femoral head. This scheme was applied: (1) to avoid the influence of the indentation site on hardness, as it has been demonstrated that hardness changes in different regions of the femoral head (Coats et al., 2003); (2) to assure a minimum distance of 3 diagonals (i.e. 120 mm since an indentation diagonal was about 40 mm) between indentations (ASTM E384). The final location of the indentation was
2.2. Specimen preparation The femoral heads obtained from total hip arthroplasty patients were immersed in 70% ethanol solution after removal (within 12 h) and kept immersed until the beginning of the sample preparation. The femoral heads obtained from IIAM were frozen at 20 1C directly after harvesting, performed within 24–96 h post-mortem (NB within 12 hours post-mortem the corpse was put in a refrigerator at 4 1C) and kept frozen during shipping. Thereafter, the samples were thawed and immersed in 70% ethanol solution. All samples were kept in ethanol Table 1 Mean age, with standard deviation and range, of the subjects included in the four groups. Group
Gender
N
Age
Age min
Age max
CG
F M F M
5 15 10 10
64 7 14 69 7 10 61 7 11 65 7 13
49 51 42 47
83 82 75 85
OAG
Fig. 1. Scheme showing the locations of the 60 indentations performed on each femoral head.
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598 defined with respect to a minimum distances between indentation-tissue edge/ pore of 2.5 diagonals (100 mm) (Johnson and Rapoff, 2007). On the polished surface of the trabecular structure, two different microstructures were found: packs of parallel-lamellae (PL, Fig. 2) and secondary osteons (SO, Fig. 2) (Lozupone and Favia, 1990; Sato et al., 1986). SO and trabecular cross sections (which are similar in appearance) were distinguished by the presence of a vessel channel at the centre of the SO. Considering the fact that indentations were performed along the main trabecular direction and perpendicular to a frontal plane, most trabeculae were cut roughly longitudinally, and trabecular cross sections were not
1595
investigated in the present study. Indentations were done on the target location despite the type of microstructure, which was registered together with the hardness value. In four cases, in which the femoral heads were too small, it was not possible to perform all the 60 indentations. Therefore, these specimens were indented only 50 times. A total of 2360 indentations were done. All indentations where one diagonal was 4 15% longer than the other were discharged, as suggested by Hodgskinson et al. (1989), leaving 2220 valid measurements.
2.4. Statistical analysis The Chauvenet criterion was applied once to all valid measurements to exclude outliers. Since numerous hardness measurements were performed on the same sample, a repeated measures ANOVA was used. Within-sample effect (i.e. indentation location or distance from joint surface) and among-samples effects (i.e. pathology, gender, and age) on hardness were investigated. Furthermore, the possible interactions among these variables were analysed. To allow the analysis, the age values were divided into three groups with 15 years intervals (41–55; 56–70; 71–85) (Wolfram et al., 2009). A w2-test was done to evaluate whether there were differences in distribution of the two structures between the groups. A multifactorial ANOVA was used to evaluate differences in hardness between the two types of structure, only considering the significant parameters highlighted in the repeated measures ANOVA.
3. Results A total of 35 hardness values, out of 2220, were excluded for the Chauvenet criterion. Average hardness values versus age and the respective regression line for the two groups, split by gender, were plotted in Fig. 4. No within-sample effect (i.e. distance from joint surface) or any interactions with the investigated parameters were found (ANOVA: p values in the range 0.34–0.99). It was found that the hardness was not dependant on gender or age (ANOVA: p¼0.98 and p¼0.13, respectively). Conversely, the hardness of the CG (33.575.9 HV) was about 13% higher (ANOVA: p o0.001) compared to the hardness of the OAG (29.376.2 HV). No interactions among investigated parameters were found (p values in the range of 0.28–0.86). A difference in distribution of the two microstructures was found between CG and OAG (w2-test: p o0.001). In fact, SO was observed in 15% of the observations in CG versus 28% in OAG. The data, grouped according to pathology and structure are reported in Fig. 5. A difference in hardness was found between the microstructures (ANOVA po0.001). In particular the PL were about 10% harder than the SO for both pathological and
Fig. 2. Examples of both the structures found during the tests. In particular both a secondary osteon (SO) and a microstructure composed by parallel-lamellae (PL) are indicated.
Fig. 3. Example of an indentation being measured on the trabecular bone tissue surface.
1596
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598
Fig. 4. Hardness values versus age grouped according to pathology and gender.
Fig. 5. Hardness values of the groups split by pathology and microstructure. Bars represent standard deviations. The sample size is indicated in each block.
non-pathological tissues. Moreover, the hardness of both microstructures of the CG (34.0 75.7 HV for PL and 30.976.6 HV for SO) was about 13% higher compared to the hardness of the OAG (30.2 75.7 HV for PL and 27.1 76.9 HV for SO). 4. Discussion The aim of this study was to evaluate whether reduced bone tissue quality, evaluated in term of hardness, is associated with severe osteoarthritis. Furthermore, it was also investigated whether age and gender of the subjects affect the hardness of trabecular tissue. In this study 20 femoral heads extracted from subjects without any bone or cartilage diseases and 20 femoral heads extracted from osteoarthritic subjects were tested by means of microindentation. In the range considered in this study (from 42 to 85 years), age was not correlated with hardness. It should be highlighted that only 40 subjects were included in this study due to difficulties in collecting bone samples, especially from non-pathological subjects. Additionally, only four specimens out of 40 were collected from subjects more than 80 years old (80, 82, 83, and 85), among
which only one female subject was included. Therefore, a slight potential decline in trabecular tissue hardness cannot be definitively excluded. However, the absence of a correlation between hardness and age (in the range 40–80) is in agreement with the study of Weaver (1966) in which it was found that, after bone maturation (around 25–30 years), the mineralisation of the interstitial tissue reaches an equilibrium and the hardness remains constant. Moreover, it has been found that the tissue elastic modulus of trabecular bone is correlated to its hardness (Hoffler et al., 2005). Considering this fact, the result of the present study is in line with the findings of Wolfram et al. (2009), which found no correlation between age and tissue elastic modulus measured in human vertebral trabecular bone tissue. No difference in hardness was found between the genders. This result is in agreement with previous studies in which indentation tests were performed on human bone tissue (Coats, et al., 2003; Hoffler et al., 2000a, 2000b; Weaver, 1966; Wolfram, et al., 2009). Both results are in line with the study of Perilli et al. (2007), in which it was shown that mechanical strength of trabecular human bone, extracted from osteoarthritic subjects, is not significantly affected by age or gender. Conversely, subjects with severe OA were found to have a reduced hardness of about 13% in the trabecular bone tissue of the femoral head, both pooling and splitting the two structures PL and SO. Hence, the different distributions of PL and SO found in the two groups (OAG and CG) did not bias the difference in hardness. This reduction in mechanical properties at the microstructural level is in line with the study of Brown et al. (2002), which found an altered matrix composition and a reduced compressive modulus in samples extracted from osteoarthritic subjects compared to normal ones with similar apparent density. Moreover, Li and Aspden (1997) found a change in the bone composition of osteoarthritic subjects and in particular an increase in water content and a decrease in mineral content compared to subjects without any cartilage or bone diseases. Higher water content and lower mineralisation might lead to a less hard tissue. Additionally, the PL had higher hardness ( + 10%) than the SO both in non-pathologic and pathologic tissue. This result might be explained by the spatial organisation of the lamellae. Furthermore, it was observed that in tissue extracted from subjects with OA the probability of indenting a SO was higher than a PL. It has been suggested that an increment of bone remodelling is seen in osteoarthritic subjects (Bailey et al., 2004; Bettica et al., 2002; Lavigne et al., 2005). Consequently, hypothetically osteoarthritic bone tissue might be composed of a larger number of younger, less mineralised and less hard structures. Therefore, the difference found in hardness of the SO and PL might also be partially due to a different degree of mineralisation of the two microstructures. However, this hypothesis should be verified with a detailed analysis of mineral content in both types of structures. The distance from the joint surface did not affect the hardness in none of the groups. This was unexpected in the OAG, considering that OA involves degeneration of the tissues of the hip joint. However, in this study only severe OA was investigated. Studying different grades of OA would be necessary to understand how the distance from the joint surface affects the bone hardness and could supply more details about the OA pathogenesis. In fact, whether all the above-mentioned changes are causes or consequences of OA is not clear, yet. There is only one study known to the authors that has compared the hardness of human trabecular bone extracted from subjects with different pathologies (Coats et al., 2003). In that study, trabecular tissues extracted from subjects affected by OA and subjects affected by osteoporosis were tested. For the osteoarthritic subjects an average of 42.275.8 HV was found, which is about 45% higher than the average value in the present
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598
study. This disagreement might be explained by the different preparations of the tested specimens. In the study of Coats et al. (2003) the tissue was embedded in acrylic resin before testing whereas in this study the specimens were tested hydrated without previous embedding, as recommended by Dall’Ara et al. (2007). The different procedures may explain a difference of about 30–40% (Dall’Ara et al., 2007; Weaver, 1966). The residual difference might be due to the different applied loads and/or the locations selected for the indentations. In that study in fact a smaller applied load was used and indentations were performed only in two small and localised areas of the femoral head, while in this study the whole head was investigated along the main trabecular direction. An indirect comparison for the CG can be obtained confronting the present results with those obtained by nanoindentation. The hardness found in the CG (33.5 HV equivalent to 0.329 GPa) seems to be in line with the hardness values of femoral neck trabecular bone, derived from the graphs reported by Zysset et al. (1999) and Hoffler et al. (2000a, 2000b). The small differences found might be due to the different anatomical sites indented and the different protocols used. The present study has four main limitations: the low number of non-pathological female subjects from whom specimens were retrieved, the unavailability of specimens from old subjects ( 480 years old), the absence of indentations on trabecular cross sections, and the absence of trabecular tissue samples collected from subject with OA at different stages. Despite these limitations the collected data showed an effect of OA on the trabecular bone of the femoral head, whereas a definitive conclusion about the effect of age on hardness would require a larger donor sample size. Additionally, the availability of tissue sample collected at different degrees of OA would allow investigating the OA pathogenesis, i.e. whether the reduced quality of the tissue is a cause or a consequence of the pathology. In conclusion considering trabecular bone tissue from the femoral head it was found that:
The tissue hardness of osteoarthritic subjects is about 13% lower than that of normal subjects.
In osteoarthritic subjects the probability to find a circular
lamellae microstructure is higher than in non-pathologic subjects. Parallel-lamellae microstructure is harder (+ 10%) than circular-lamellae microstructure for both osteoarthritic and nonpathological subjects. Hardness is not dependent on gender. A larger donor sample size would be necessary to definitively exclude the existence of an effect of age.
Conflict of interest statement None declared.
Acknowledgments This work was partially supported by the European Community (project number: IST-2006-035763; project title: 3D Anatomical Human and project number; IST-2004-026932; project title: Living Human Digital Library; acronym: LHDL). The authors wish to thank Barbara Bordini for the statistical analysis and Luigi Lena for the illustrations.
1597
References Bailey, A.J., Mansell, J.P., Sims, T.J., Banse, X., 2004. Biochemical and mechanical properties of subchondral bone in osteoarthritis. Biorheology 41, 349–358. Bettica, P., Cline, G., Hart, D.J., Meyer, J., Spector, T.D., 2002. Evidence for increased bone resorption in patients with progressive knee osteoarthritis: longitudinal results from the Chingford study. Arthritis and Rheumatism 46, 3178–3184. Bouxsein, M.L., 2003. Bone quality: where do we go from here? Osteoporosis International 14, S118–S127. Brown, S.J., Pollintine, P., Powell, D.E., Davie, M.W., Sharp, C.A., 2002. Regional differences in mechanical and material properties of femoral head cancellous bone in health and osteoarthritis. Calcified Tissue International 71, 227–234. Coats, A.M., Zioupos, P., Aspden, R.M., 2003. Material properties of subchondral bone from patients with osteoporosis or osteoarthritis by microindentation testing and electron probe microanalysis. Calcified Tissue International 73, 66–71. Cooper, C., Cook, P.L., Osmond, C., Fisher, L., Cawley, M.I., 1991. Osteoarthritis of the hip and osteoporosis of the proximal femur. Annals of the Rheumatic Diseases 50, 540–542. Currey, J.D., 2003. Role of collagen and other organics in the mechanical properties of bone. Osteoporosis International 14 (Suppl. 5), 29–36. Dall’Ara, E., Ohman, C., Baleani, M., Viceconti, M., 2007. The effect of tissue condition and applied load on Vickers hardness of human trabecular bone. Journal of Biomechanics 40, 3267–3270. Evans, F.G., Behiri, J.C., Currey, J.D., Bonfield, W., 1990. Microhardness and Young’s modulus in cortical bone exhibiting a wide range of mineral volume fractions, and in a bone analogue. Journal of Materials Science: Materials in Medicine 1, 38–43. Fazzalari, N.L., Parkinson, I.H., 1998. Femoral trabecular bone of osteoarthritic and normal subjects in an age and sex matched group. Osteoarthritis and Cartilage 6, 377–382. Fazzalari, N.L., Vernon-Roberts, B., Darracott, J., 1987. Osteoarthritis of the hip. Possible protective and causative roles of trabecular microfractures in the head of the femur. Clinical Orthopaedics and Related Research 216, 224–233. Fratzl, P., Gupta, H.S., Paschalis, E.P., Roschger, P., 2004. Structure and mechanical quality of the collagen–mineral nano-composite in bone. Journal of Materials Chemistry 14, 2115–2123. Hengsberger, S., Kulik, A., Zysset, P., 2002. Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. Bone 30, 178–184. Hodgskinson, R., Currey, J.D., Evans, G.P., 1989. Hardness, an indicator of the mechanical competence of cancellous bone. Journal of Orthopaedic Research 7, 754–758. Hoffler, C.E., Guo, X.E., Zysset, P.K., Goldstein, S.A., 2005. An application of nanoindentation technique to measure bone tissue Lamellae properties. Journal of Biomechanical Engineering 127, 1046–1053. Hoffler, C.E., Moore, K.E., Kozloff, K., Zysset, P.K., Goldstein, S.A., 2000a. Age, gender, and bone lamellae elastic moduli. Journal of Orthopaedic Research 18, 432–437. Hoffler, C.E., Moore, K.E., Kozloff, K., Zysset, P.K., Brown, M.B., Goldstein, S.A., 2000b. Heterogeneity of bone lamellar-level elastic moduli. Bone 26, 603–609. Huja, S.S., Katona, T.R., Moore, B.K., Roberts, W.E., 1998. Microhardness and anisotropy of the vital osseous interface and endosseous implant supporting bone. Journal of Orthopaedic Research 16, 54–60. Johnson, W.M., Rapoff, A.J., 2007. Microindentation in bone: Hardness variation with five independent variables. Journal of Materials Science: Materials in Medicine 18, 591–597. Lavigne, P., Benderdour, M., Lajeunesse, D., Reboul, P., Shi, Q., Pelletier, J.P., MartelPelletier, J., Fernandes, J.C., 2005. Subchondral and trabecular bone metabolism regulation in canine experimental knee osteoarthritis. Osteoarthritis Cartilage 13, 310–317. Li, B., Aspden, R.M., 1997. Composition and mechanical properties of cancellous bone from the femoral head of patients with osteoporosis or osteoarthritis. Journal of Bone and Mineral Reasearch 12, 641–651. Linde, F., Sorensen, H.C., 1993. The effect of different storage methods on the mechanical properties of trabecular bone. Journal of Biomechanics 26, 1249–1252. Lozupone, E., Favia, A., 1990. The structure of the trabeculae of cancellous bone. 2. Long bones and mastoid. Calcified Tissue International 46, 367–372. Mansell, J.P., Bailey, A.J., 1998. Abnormal cancellous bone collagen metabolism in osteoarthritis. Journal of Clinical Investigation 101, 1596–1603. Mansell, J.P., Tarlton, J.F., Bailey, A.J., 1997. Biochemical evidence for altered subchondral bone collagen metabolism in osteoarthritis of the hip. British Journal of Rheumatology 36, 16–19. Neilson, M., White, A., Malik, U., Morrison, E., McGill, P.E., McDonald, S.W., 2004. Changes in bone architecture in the femoral head and neck in osteoarthritis. Clinical Anatomy 17, 378–391. Ohman, C., Baleani, M., Perilli, E., Dall’Ara, E., Tassani, S., Baruffaldi, F., Viceconti, M., 2007. Mechanical testing of cancellous bone from the femoral head: experimental errors due to off-axis measurements. Journal of Biomechanics 40, 2426–2433. Perilli, E., Baleani, M., Ohman, C., Baruffaldi, F., Viceconti, M., 2007. Structural parameters and mechanical strength of cancellous bone in the femoral head in osteoarthritis do not depend on age. Bone 41, 760–768. Phelps, J.B., Hubbard, G.B., Wang, X., Agrawal, C.M., 2000. Microstructural heterogeneity and the fracture toughness of bone. Journal of Biomedical Materials Research 51, 735–741.
1598
E. Dall’Ara et al. / Journal of Biomechanics 44 (2011) 1593–1598
Radin, E.L., Paul, I.L., Tolkoff, M.J., 1970. Subchondral bone changes in patients with early degenerative joint disease. Arthritis and Rheumatism 13, 400–405. Radin, E.R., Paul, I.L., Rose, R.M., 1972. Pathogenesis of primary osteoarthritis. Lancet 1, 1395–1396. Rho, J.Y., Tsui, T.Y., Pharr, G.M., 1997. Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation. Biomaterials 18, 1325–1330. Sato, K., Wakamatsu, E., Sato, T., Honma, T., Kotake, H., Byers, P.D., 1986. Histomorphometric study of trabecular channels in normal iliac bone. Calcified Tissue International 39, 2–7. Stea, S., Visentin, M., Savarino, L., Ciapetti, G., Donati, M.E., Moroni, A., Caja, V., Pizzoferrato, A., 1995. Microhardness of bone at the interface with ceramic-coated metal implants. Journal of Biomedical Materials Research 29, 695–699.
Weaver, J.K., 1966. The microscopic hardness of bone. Journal of Bone and Joint Surgery. American Volume 48, 273–288. Wolfram, U., Wilke, H.J., Zysset, P.K., 2009. Rehydration of vertebral trabecular bone: influences on its anisotropy, its stiffness and the indentation work with a view to age, gender and vertebral level. Bone 46, 348–354. Zioupos, P., Currey, J.D., Casinos, A., 2000. Exploring the effects of hypermineralisation in bone tissue by using an extreme biological example. Connective Tissue Research 41, 229–248. Zysset, P.K., 2009. Indentation of bone tissue: a short review. Osteoporosis International 20, 1049–1055. Zysset, P.K., Guo, X.E., Hoffler, C.E., Moore, K.E., Goldstein, S.A., 1999. Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur. Journal of Biomechanics 32, 1005–1012.