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Bone microarchitecture evaluated by histomorphometry
Micron 36 (2005) 609–616 www.elsevier.com/locate/micron
Review
Bone microarchitecture evaluated by histomorphometry L. Dalle Carbonarea,*, M.T. Valentia, F. Bertoldoa, M. Zanattaa, S. Zenaria, G. Realdib, V. Lo Cascioa, S. Gianninib a
Department of Biomedical and Surgical Sciences, Medicina Interna D, University of Verona, Italy b Department of Medical and Surgical Sciences, Clinica Medica I, University of Padova, Italy Received 15 May 2005; revised 4 July 2005; accepted 5 July 2005
Abstract The increasing use of densitometric devices for assessing bone fragility has progressively strengthened the assumption that mass is the most important property determining bone mechanical competence. Nevertheless, structure and microarchitecture are relevant aspects of bone strength. The study of microarchitecture is based on the measure of width, number, and separation of trabeculae as well as on their spatial organization. There are several methods to assess bone architecture, particularly at the trabecular level. In particular, histomorphometry, based on the use of optical microscopy and on the principles of quantitative histology and stereology, evaluates microarchitecture two-dimensionally, even if these measures appear well correlated to the three-dimensional structure and properties of bone. In addition, new computerized methods allow the acquisition of more sophisticated measurements by means of a digitizer have been introduced to integrate the use of the microscope. These methods supply information on trabecular width as well as on its distribution and on the organization of the trabeculae in the marrow space. Microarchitecture seems to be a determinant of bone fragility independent of bone density and it is important for understanding the mechanisms of bone fragility as well as the action of the drugs used to prevent osteoporotic fractures. Several in vivo studies (on animals and humans) can provide an additional interpretation for the anti-fracture effect of such drugs. For instance, bisphosphonates and parathyroid hormone seem to preserve or even improve microarchitecture. The challenge for the future will be to evaluate bone quality in vivo with the same or better resolution and accuracy than the invasive methods used today. q 2005 Elsevier Ltd. All rights reserved. Keywords: Bone density (BMD); Bone strength; Connectivity; Fractal dimension (D); Fractures; Histomorphometry; Microarchitecture; Marrow star volume (MSV); Strut analysis
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods for the assessment of microarchitecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histomorphometric approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microarchitecture and fractures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microarchitecture and treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations used B.Ar, Bone area; T.Ar, Tissue area; Tb.Ar, Trabecular area; B.Pm, Bone perimeter; BV/TV, Bone volume; Tb.N, Trabecular number; Tb.Th, Trabecular thickness; Tb.Sp, Trabecular separation; MSV, Marrow star volume; ICI, Interconnectivity index; TBPf, Trabecular bone pattern factor; D, Fractal analysis; E/TV, Euler number/tissue volume; QCT, Quantitative computed tomography; hrCT, High resolution computed tomography; vQCT, Volumetric quantitative computed tomography; hrMR, High resolution magnetic resonance imaging. * Corresponding author. Tel.: C390 45 8074684; fax: C390 45 583041. E-mail address: [email protected] (L. Dalle Carbonare). 0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2005.07.007
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1. Introduction The increasing use of densitometric devices for assessing bone fragility has progressively strengthened the assumption that mass is the most important property determining bone mechanical competence. In the last decade, Dual Energy X-ray Absorptiometry (DXA) has been considered the most valuable noninvasive method for measuring bone mineral density (BMD). However, many recent observations indicate that bone strength is only partially explained by bone density. Bone volume alone accounted for only 76% of the variability in strength, whereas a combination of bone volume and several architectural features explained up to 90% of the strength variability (Dempster, 2003). Bone shape and internal structure are influenced by load and different stimuli and stresses, which represent the result of both, muscular tension and gravity. On the other hand, the interaction between the genetic elements and the environmental stimuli lead to a structure that can provide the best resistance to load, stress or compression, on account of the spatial orientation of the trabecular network as well. Bone shape is fashioned into three-dimensional geometric and architectural masterpieces of biomechanical engineering—minimal mass optimized in size and shape according to whether the main function is as a lever or a spring. For load bearing and leverage, the need for stiffness is favored over flexibility by the fashioning of mineralized tissue into long bones with a marrow cavity displacing the mineralized cortex distant from neutral bone axis. Vertebral bodies, spring-like shock absorbers on which stiffness is sacrificed for flexibility, show an open-celled porous cancellous structure able to deform and return to its original size and shape without cracking (Seeman, 2003). The structural determinants of bone mechanical strength include width and porosity in the cortical bone; shape, width, connectivity, and anisotropy in the trabecular bone. It has been observed that during age-related bone involution the loss of entire trabeculae would result in higher loads and, consequently, in the thickeing of the remaining elements. In particular, the number of horizontal trabeculae decreases throughout life, whereas vertical trabeculae are resorbed more slowly and tend to increase in width with age (Atkinson, 1967). This is probably due to the fact that the load in vertebral cancellous bone is mainly placed on the vertical elements, thus the compensatory hypertrophy or thickening effect is only expected in the vertical trabeculae. The strength of a vertical trabecula is inversely proportional to the square power of its effective length. This means that a loss of a single horizontal strut or cross-tie increases the effective length of a vertical trabecula by a factor of two, but it will reduce its compressive strength by a factor of four. (Atkinson, 1967). Based on biomechanical characteristics reported above, the evaluation and quantification of these specific properties of bone could provide important information on the fragility status of the skeleton.
From another point of view, it has also been observed (Chesnut III and Rosen, 2001) that the increases in bone mass recorded after drug therapies for osteoporosis can just partly explain the decrease in fracture incidence after these treatments. Therefore, the effect of some of these therapies on microarchitecture could explain their efficacy and rapidity of action. Consequently, there is a growing interest in the quantitative assessment of this particular morphological aspect of bone.
2. Methods for the assessment of microarchitecture The study of microarchitecture is based on the measure of width, number, and separation of trabeculae as well as on their spatial organization. Table 1 shows the list of the main parameters of microarchitecture, which are explained in the next section. The trabecular network, defined as connectivity, is a three-dimensional property that describes the typology of the various connections between the so-called nodes (the structural units that represent the confluence of three or more trabeculae) and the connecting segments (struts and termini). From a quantitative standpoint, connectivity is an index of the variety of links among the trabeculae and can be defined as the major number of connections that can be broken before structure integrity is completely lost (DeHoff et al., 1972). There are several methods to assess bone architecture, particularly at the trabecular level, that is, to detect the organization of bone in space and to measure the complexity of its structure quantitatively. Two different approaches can be identified. (A). The first uses optical microscopy and is based on the principles of histomorphometry (namely, quantitative histology based on stereology), which evaluates microarchitecture on two-dimensional bone sections (Parfitt, 1983). In addition, new stereological methods Table 1 Main histomorphometric parameters of microarchitecture Parameter
Abbreviation
Units
Bone Volume/Tissue Volume (%) Trabecular Number Trabecular Thickness Trabecular Separation Total Skeletal Length Node Number/Tissue Volume Node/Termini Marrow Star Volume Fractal Dimension Trabecular Bone Pattern Factor Interconnectivity Index Euler Number
BV/TV
%
Tb.N Tb.Th Tb.Sp TSL NN/TV N/T MaSV D TBPf
/mm mm mm mm N/mm2 % N/mm3 – –
ICI E/TV
– /mm2
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are now available to provide valuable information on the connectivity of trabecular bone and network complexity (Garrahan et al., 1986). (B). The second applies the most modern diagnostic techniques. Noninvasive physical techniques such as QCT (quantitative computed tomography), especially high resolution computed tomography (hrCT) at 100– 400 mm, volumetric quantitative computed tomography (vQCT) and high resolution magnetic resonance imaging (hrMR) at 100–200 mm have been proposed for the assessment of microarchitecture (Feldkamp et al., 1989; Muller et al., 1998; Laib et al., 2002). The last approaches are interesting and promising but, at this moment, their use is limited because they provide only approximations of micro-structural parameters, with considerable threshold and resolution dependence. Furthermore, some of these approaches are limited by the high exposure to ionizing radiation, and by the expense and limited availability of the equipment. Ultrasound measurements have also been proposed to investigate bone quality, but the poor understanding of what components are measured by broadband attenuation and speed of sound limit their use in this setting (Chappard et al., 1999).
3. Histomorphometric approach The first approaches to quantitative evaluation of trabecular bone structure were based on direct and indirect measure of trabecular width, separation, and number (Wakamatsu and Sisson, 1969; Whitehouse, 1974; Aaron et al., 1987). The measurement of trabecular parameters on the bone sections was initially obtained using a microscope with an ocular equipped with a special grid, such as those shown in Fig. 1. The Trabecular Number is defined as (BV/TV)/Tb.Th, where BV is Bone Volume, TV is the volume of the examined tissue, and Tb.Th is the thickness of the trabecula. The Trabecular Bone Volume (BV/TV) can be also obtained from measurements of bone area (B.Ar)
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and cancellous tissue area (BV/TVZ100!B.Ar/T.Ar). In this approach, Trabecular Thickness, Number, and Separation are derived from trabecular area and perimeter (B.Ar, B.Pm), applying these algorithms: Trabecular Thickness (Tb.ThZ1.199!B.Ar/2/B.Pm; the factor 1.199 is used to correct section obliquity) Trabecular Number (Tb.NZ Tb.Ar!10/Tb.Th, expressed in mm); Trabecular Separation (Tb.Sp: 1000/Tb.N–Tb.Th, expressed in mm) (Chappard et al., 1999). These parameters describe the basic relationship between space and trabecular network. Conventionally, all these parameters are expressed as volume instead of area, even if they are evaluated in twodimensional sections, because they offer an inferred estimation of the spatial organization of the trabecular network. Similarly, the Trabecular Separation, defined as the distance between the edges of the trabeculae, is expressed in three-dimensional units (Parfitt et al., 1987). More recently, new computerized methods that allow the acquisition of more sophisticated measurements by means of a digitizer have been introduced to integrate the use of the microscope (Clermonts and Birkennha¨ger-Frenkel, 1985; Garrahan et al., 1987). These methods supply information on trabecular width as well as on its distribution and on the organization of the different trabeculae in a bone section. In particular, through the process of ‘skeletonization’ of the trabecular bone (that is the measurement of the trabecular profiles and the count of their connections on twodimensional sections by a computed analysis, the so called ‘strut analysis’), it is possible to obtain a network of twodimensional components (the struts), which reproduces the trabecular distribution in space, the so called ‘Trabecular network’ (Garrahan et al., 1986, Fig. 2). The link between three or more struts constitutes a node. When one side of a strut is not joined to a node it is referred to as ‘free end’ or ‘termini’, which represents an interruption in the trabecular network (Parfitt, 1986; Day et al., 2004). The ratio between nodes and termini in a section is an index of spatial connectivity. Other parameters that allow an indirect evaluation of the trabecular connectivity, through the assessment of the marrow connectivity, are:
Fig. 1. The direct measurement of trabecular parameters can be obtained by using an ocular equipped with a special grid. The two panels represent two different grid used for these measurements.
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the mean length of the arrows (three-dimensionally the mean volume) between a random point and a trabecular boundary is higher than the same parameter in the right panel. This value is an evaluation of marrow cavity extension. Consequently, the higher the value, the poorer the trabecular connectivity. Trabecular Bone Pattern Factor (TBPf): is the relation between convex and concave elements (Fig. 4). The rationale of this technique is supported by the fact that in well-connected structure the concave surfaces are abundant, while the convex are more numerous in disconnected structure (Hahn et al., 1992). So, we can consider a concave element as the expression of a wellconnected structure (Chappard et al., 1999; Vogel et al., 1990). In Fig. 4, A is a depiction of a convex figure, while B is a concave structure. When trabecular network is highly disconnected, the convex surfaces (A) are predominant and a dilatation process increases drastically the perimeter, while area is only modestly affected. Opposite trend is observed after a dilatation process of a concave structure. TBPf is defined as: TBPfZ(bone perimeter–bone perimeter after dilatation)/(bone area– bone area after dilatation). Consequently, TBPf is low in well-connected bone and high when highly disconnected trabeculae are observed (Hahn et al., 1992; Chappard et al., 1999). Index of Interconnectivity (ICI): is the connectivity of the marrow cavities which can be evaluated after skeletonization (see above). Terminal ends and the branching nodes of the skeleton are identified and shortest branches are eliminated. Then, the total number of nodes (N), node-to-node branches (NN) and node-to-free
Fig. 2. The strut analysis. The measurement of the trabecular profiles and their connections on two-dimensional sections by a computed analysis (skeletonization) convert the trabecular structure in a network of linear components. The orange points represent the nodes, that is, the connection between three or more struts (segment representing a trabecula, white points). The green points represent the free-ends or termini, which are broken or interrupted elements of trabecular network (For interpretation of the reference to colour in this legend, the reader is referred to the web version of this article).
Marrow Star Volume (MSV): is defined as the mean volume of all the parts of an object when seen unobscured along uninterrupted straight lines in all directions from random points inside the object (Vesterby et al., 1989). In cancellous bone, MSV provides an estimate of the mean size of the marrow space in three-dimensions and thus reflects connectivity. Fig. 3 shows a representation of MSV: in the left panel
A
B
Fig. 3. Schematic drawing to illustrate the concept of the Marrow Star Volume (MSV). This parameter represents the mean volume of all the parts of an object that can be unobscured in all directions from a point inside the object. By the analysis of marrow space distribution, we obtain an indirect evaluation of the trabecular network organization. The higher the mean of the segments the lower the trabecular connectivity. The panel A shows a higher value of MSV (obtained from the mean of the arrow length) than the panel B, highlighting reduced trabecular connectivity.
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A
B
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A
B
C
D
Branches of connection
Fig. 4. Representation of Trabecular Bone Pattern Factor (TBPf). The rationale of TBPf is that in well-connected structure the concave surfaces are abundant, while the convex surfaces are more numerous in disconnected structure. So, we can consider a concave element as the expression of a well-connected structure. When trabecular network is highly disconnected, the convex surfaces are predominant and a dilatation process increases the perimeter of the structure, while area is only modestly affected. The trend is opposite in a well-connected structure. A: Spherical (convex) figureZpoor connected structure; B: figure with concave elementsZwell-connected structure.
end branches (NF) are determined. The number of tree (T) is also obtained (a tree is an independent portion of the medullary space totally enclosed by a trabecular structure). The Index of interconnectivity of the bone marrow is then defined as (N!NN)/[T!(NFC1)]. The higher the interconnectivity of the marrow (characterized by an elevated number of nodes and segmental branches and few trees), the higher the ICI and the fragmentation of the trabecular network (Le et al., 1992). Euler Number (E): expressed per tissue volume (E/TV), i.e. the number of holes minus the number of connected components, which can be interpreted as the maximum number of branches that could be removed without breaking the network into different parts (Feldkamp et al., 1989). Total number of trabecular profiles is indicated with n, while the number of marrow cavities with m. E Z nKm Low value of E indicates a more connected bone. Negative values are obtained from high-connected structure (Chappard et al., 1999). ‘fractal’ analysis or Dimension (D): describes how an object fills space with relation to its structure (Weinsteinm and Majumdar, 1994). The fractal dimension of the trabecular network can be measured by ‘box counting method’ applied on the trabecular image (Fig. 5). A sort of chess board, a grid of boxes, 3, of different sizes is superimposed on the perimeter to be characterized, and the number of boxes that contain the boundary points of the cancellous bone (trabe-
Fig. 5. Representation of Fractal Analysis or Dimension (D). In the boxcounting technique, a grid of boxes, 3, of different sizes is superimposed on the perimeter, and the number of boxes that contain the boundary points of the cancellous bone (trabeculae), N(3), is counted. Based on the graph log N(3) as a function of log 3, the Fractal Dimension (D) can be calculated from the negative slope of the curve. This is an example of the variation in N(3) for a sample (red square) of four different box sizes. A: N(3)Z1; B: N(3)Z4; C: N(3)Z8; D: N(3)Z18 (For interpretation of the reference to colour in this legend, the reader is referred to the web version of this article) .
culae), N(3), are counted. In Fig. 5, an example of the variation in N(3) for a sample (red square) of four different box sizes is shown. The Fractal Dimension (D) is calculated as the negative slope of the linear portion of the curve obtained by plotting the logarithm of N(3) and 3. This parameter represents the smallest number of boxes of side 3 required to cover completely the trabecular boundaries and indicates the perimeter examined with a 3 scale ratio. Each of these parameters gives a distinctive analysis of trabecular bone architecture. This approach is limited by the necessity to infer three-dimensional information from a two-dimensional evaluation. Nevertheless, there are several lines of evidence confirming that measurements on two-dimensional sections are well correlated to the three-dimensional structure and properties of bone (Feldkamp et al., 1989).
4. Microarchitecture and fractures It is well known that exists a direct correlation between Bone Mineral Density (BMD) at the spine and femur and the
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to be a mass-indipendent technique in the evaluation of bone fragility and fracture risk assessment.
risk fracture at these skeletal sites, but it has been also observed a wide overlap in the BMD of patients with and without fractures. As already explained, bone strength is not determined only by mass and BMD. Nevertheless, the combination of many factors can predict more accurately the occurrence of new fractures. With regard to the relationship between microarchitecture and fractures, recent observations seem to confirm that microstructural alterations are important determinants of bone fragility, independently of bone density (Audran et al., 2001; Hordon et al., 2000; Reeker, 1993; Legrand et al., 2000). In the first of these studies (on orchidectomized rats) the evaluation of microarchitectural parameters such as Marrow Star Volume, Fractal Dimension, and node count were dramatically affected. This confirms that bone loss and fragility secondary to orchidectomy was associated with trabecular perforation and reduction of the complexity of trabecular network (Audran et al., 2001). In the other two-dimensional histologic study (Hordon et al., 2000), women with fractures tended to have lower bone surface (BS), wider and fewer trabeculae (Tb.Le and Strut number, respectively), lower mean trabecular plate density (MTPD; known as trabecular number/mm, or Tb.N/mm), fewer trabecular nodes, more termini, wider Trabecular Separation, larger Marrow Star Volume and Trabecular Bone Pattern Factor. In other similar studies significant differences were found in number and thickness of trabecular plates, which were fewer and more widely separated, in Marrow Star Volume (MSV), greater in fracture subject, and number of trabeculae, nodes and free-ends (Hordon et al., 2000; Reeker, 1993). All these data are consistent with loss of connectivity in fracture subjects. Finally, in men affected by idiopathic osteoporosis, Legrand et al. (2000) observed that the differences in bone microarchitecture parameters with the same mass and bone volume were the only discriminating factor between patients with or without vertebral fracture. In conclusion, the histological approach permits us to study accurately bone microarchitecture and demonstrates
A
5. Microarchitecture and treatment Bone microarchitecture seems to be important for understanding the mechanisms of bone fragility as well as the action of the drugs used to prevent osteoporotic fractures. The exact mechanism of the anti-fracture effect of drugs inhibiting resorption has not been completely explained, but increases in bone mass alone cannot account for their efficacy. More attention has lately been paid to the microarchitecture evaluation as a peculiar feature of bone quality, which is one of the targets of the drugs currently in use, such as antiresorptive treatment (bisphosphonates), selective estrogen receptor modulators (SERM, raloxifene) and anabolic agents (teriparatide). For instance, a histomorphometric study on patients undergoing 3-year therapy with alendronate showed that bone lamellar architecture was preserved with no qualitative alteration after treatment (Chavassieux et al., 1997). In a recent study, we found significant differences in terms of microarchitecture in rats treated with glucocorticoids alone or with risedronate (unpublished data, Fig. 6). The administration of risedronate preserved microarchitecture, expressed as direct (NdN/TV, Nd/Tm) and indirect parameters (MSV) with respect to rats treated with glucocorticoids alone. The studies on the use of parathyroid hormone (PTH, teriparatide) also confirm its action on microarchitecture, which can provide an interpretation for its anti-fracture effect from a structural viewpoint (Bradbeer et al., 1992; Dempster et al., 2001). This is even more important if we consider that effects of teriparatide treatment on BMD are controversial. As a matter of fact, after teriparatide a significant increased lumbar BMD and reduced appendicular BMD were observed (Slovik et al., 1986; Hodsman et al., 1997; Lindsay et al., 1997). Nevertheless, few studies have examined the effects of
B
Fig. 6. Different microarchitectural patterns in rats treated with glucorticoids without (A) and with a bisphosphonate (risedronate) (B).
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PTH on bone histology. In a histomorphometric study, biopsy analysis revealed that women treated with teriparatide showed an increased trabecular and cortical thickness, increased BV/TV, with a reduction of MSV (Jang et al., 2003). In these subjects there was evidence of an anabolic effect particularly on the endosteal surface, where an increase in the packet wall width was observed (Dempster et al., 2001). This anabolic effect on cortical bone result to an increased cortical diameter, reduced marrow diameter and, consequently, in a consistent improvement of mechanical strength (Zanchetta et al., 2003). In addition, the evaluation of microarchitecture in rats treated for a long time with PTH showed that its skeletal effects are a complex function of dose and duration. On the other hand, in the rat model, short-term treatment seems to be more effective than near-life treatment, because PTH stimulates skeletal growth throughout life, resulting in abnormal architecture (Sato et al., 2002). Further studies will be necessary to confirm and investigate these acquisitions, in order to understand the exact mechanisms leading to bone fragility and find new and more effective therapeutic strategies to face consequences of osteoporosis and other metabolic bone diseases.
6. Conclusions Microarchitecture is an important element of bone quality and its integrity contributes to the bone mechanical competence. The assessment of microarchitecture may be useful to evaluate the risk of fracture as well as the drug mechanism of action. In the last decade several methods, from the traditional quantitative histology, improved by computed analyses, to the recent applications of computed tomography and magnetic resonance, have been devised and applied to assess this specific bone property. The challenge for the future will be to evaluate bone quality in vivo with the same or better resolution and accuracy than the invasive methods in use today. This goal would be essential to introduce the evaluation of such an important determinant of bone strength as microarchitecture in the routine diagnostic approach to skeletal diseases inducing fragility.
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Le, H.M., Holmes, R.E., Shors, E.C., Rosenstein, D.A., 1992. Computerized quantitative analysis of the interconnectivity of porous biomaterials. Acta Stereologica 11 (1), 267–272. Legrand, E., Chappard, D., Pascaretti, C., Duquenne, M., Krebs, S., Rohmer, V., Basle, M.F., Audran, M., 2000. Trabecular bone microarchitecture, bone mineral density, and vertebral fractures in male osteoporosis. Journal of Bone and Mineral Research 15, 13–19. Lindsay, R., Nieves, J., Formica, C., Henneman, E., Woelfert, L., Shen, V., Dempster, D., Cosman, F., 1997. Randomized control study of effect of parathyroid hormone of vertebral-bone mass and fracture incidence among postmenopausal women on oestrogen with osteoporosis. Lancet 350, 550–555. Muller, R., Campenhout, H., Van Damme, B., Van der Perre, G., Dequeker, J., Ildebrand, T., Ruegsegger, P., 1998. Morphometric analysis of human bone biopsies: A quantitative structural comparison of histological sections and micro-computed tomography. Bone 23 (1), 59–66. Parfitt, A.M., 1983. Bone histomorphometry: techniques and interpretation. In: Recker, R.R (Ed.), The Physiologic and Clinical Significance of Bone Histomorphometric Data. CRC Press, Boca Raton, FL, USA, pp. 143–224. Parfitt, A.M., 1986. Remodeling and microstructure of bone: Relation to prevent of age related fracture. In: Vagenakis, A., Soucacos, P., Avramides, A., Segre, G., Deftos, L. (Eds.), Second International Conference on Osteoporosis: Social and Clinical Aspects. Masson Italia Editori SIA, Milano, pp. 197–209. Parfitt, A.M., Drezner, M.K., Glorieux, F.H., Kanis, J.A., Malluche, H., Meunier, P.J., Ott, S.M., Recker, R.R., 1987. Bone histomorphometry: standardization of nomenclature, symbols, and units. Journal of Bone and Mineral Research 2, 595–610.
Reeker, R.R., 1993. Architecture and vertebral fracture. Calcified Tissue International 53 (1), S139–S142. Sato, M., Vahle, J., Schmidt, A., Westmore, M., Smith, S., Rowley, E., Ma, L.Y., 2002. Abnormal bone microarchitecture and biomechanical properties with near-lifetime treatment of rats with PTH. Endocrinology 143, 3230–3242. Seeman, E., 2003. Bone quality. Osteoporosis International 14 (5), S3–S7. Slovik, D.M., Rosenthal, D.I., Doppelt, S.H., Potts Jr.., J.T., Daly, M.A., Campbell, J.A., Neer, R.M., 1986. Restoration of spinal bone in osteoporotic men by treatment with human parathyroid hormone (1-34) and 1,25-dihydroxyvitamin D. Journal of Bone and Mineral Research 1, 377–381. Vesterby, A., Gundersen, H.J.G., Melsen, F., 1989. Star volume of marrow space and trabeculae of the first lumbar vertebra: Sampling efficiency and biological variation. Bone 10, 7–13. Vogel, M., Hahn, M., Caselitz, P., Woggan, J., Pompesius-Kempa, M., Delling, G., 1990. Comparison of trabecular bone structure in man today and an ancient population in Western Germany. In: Takahashi, H.E. (Ed.), Bone Morphometry. Nishimura Co., Ltd, Niigata, Japan, pp. 220–223. Wakamatsu, E., Sisson, H.A., 1969. The cancellous bone of the iliac crest. Calcified Tissue Research 4, 147–161. Weinsteinm, R.S., Majumdar, S., 1994. Fractal geometry and vertebral compression fractures. Journal of Bone and Mineral Research 9, 1797– 1802. Whitehouse, W.J., 1974. The quantitative morphology of anisotropic trabecular bone. Journal of Microscopy 101, 153–168. Zanchetta, J.R., Bogado, C.E., Ferretti, J.L., Wang, O., Wilson, M.G., Sato, M., Gaich, G.A., Dalsky, G.P., Myers, S.L., 2003. Effects of teriparatide [recombinant human parathyroid hormone (1–34)] on cortical bone in postmenopausal women with osteoporosis. Journal of Bone and Mineral Research 18, 539–543.
Review
Bone microarchitecture evaluated by histomorphometry L. Dalle Carbonarea,*, M.T. Valentia, F. Bertoldoa, M. Zanattaa, S. Zenaria, G. Realdib, V. Lo Cascioa, S. Gianninib a
Department of Biomedical and Surgical Sciences, Medicina Interna D, University of Verona, Italy b Department of Medical and Surgical Sciences, Clinica Medica I, University of Padova, Italy Received 15 May 2005; revised 4 July 2005; accepted 5 July 2005
Abstract The increasing use of densitometric devices for assessing bone fragility has progressively strengthened the assumption that mass is the most important property determining bone mechanical competence. Nevertheless, structure and microarchitecture are relevant aspects of bone strength. The study of microarchitecture is based on the measure of width, number, and separation of trabeculae as well as on their spatial organization. There are several methods to assess bone architecture, particularly at the trabecular level. In particular, histomorphometry, based on the use of optical microscopy and on the principles of quantitative histology and stereology, evaluates microarchitecture two-dimensionally, even if these measures appear well correlated to the three-dimensional structure and properties of bone. In addition, new computerized methods allow the acquisition of more sophisticated measurements by means of a digitizer have been introduced to integrate the use of the microscope. These methods supply information on trabecular width as well as on its distribution and on the organization of the trabeculae in the marrow space. Microarchitecture seems to be a determinant of bone fragility independent of bone density and it is important for understanding the mechanisms of bone fragility as well as the action of the drugs used to prevent osteoporotic fractures. Several in vivo studies (on animals and humans) can provide an additional interpretation for the anti-fracture effect of such drugs. For instance, bisphosphonates and parathyroid hormone seem to preserve or even improve microarchitecture. The challenge for the future will be to evaluate bone quality in vivo with the same or better resolution and accuracy than the invasive methods used today. q 2005 Elsevier Ltd. All rights reserved. Keywords: Bone density (BMD); Bone strength; Connectivity; Fractal dimension (D); Fractures; Histomorphometry; Microarchitecture; Marrow star volume (MSV); Strut analysis
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods for the assessment of microarchitecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histomorphometric approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microarchitecture and fractures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microarchitecture and treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
610 610 611 613 614 615 615
Abbreviations used B.Ar, Bone area; T.Ar, Tissue area; Tb.Ar, Trabecular area; B.Pm, Bone perimeter; BV/TV, Bone volume; Tb.N, Trabecular number; Tb.Th, Trabecular thickness; Tb.Sp, Trabecular separation; MSV, Marrow star volume; ICI, Interconnectivity index; TBPf, Trabecular bone pattern factor; D, Fractal analysis; E/TV, Euler number/tissue volume; QCT, Quantitative computed tomography; hrCT, High resolution computed tomography; vQCT, Volumetric quantitative computed tomography; hrMR, High resolution magnetic resonance imaging. * Corresponding author. Tel.: C390 45 8074684; fax: C390 45 583041. E-mail address: [email protected] (L. Dalle Carbonare). 0968-4328/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2005.07.007
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1. Introduction The increasing use of densitometric devices for assessing bone fragility has progressively strengthened the assumption that mass is the most important property determining bone mechanical competence. In the last decade, Dual Energy X-ray Absorptiometry (DXA) has been considered the most valuable noninvasive method for measuring bone mineral density (BMD). However, many recent observations indicate that bone strength is only partially explained by bone density. Bone volume alone accounted for only 76% of the variability in strength, whereas a combination of bone volume and several architectural features explained up to 90% of the strength variability (Dempster, 2003). Bone shape and internal structure are influenced by load and different stimuli and stresses, which represent the result of both, muscular tension and gravity. On the other hand, the interaction between the genetic elements and the environmental stimuli lead to a structure that can provide the best resistance to load, stress or compression, on account of the spatial orientation of the trabecular network as well. Bone shape is fashioned into three-dimensional geometric and architectural masterpieces of biomechanical engineering—minimal mass optimized in size and shape according to whether the main function is as a lever or a spring. For load bearing and leverage, the need for stiffness is favored over flexibility by the fashioning of mineralized tissue into long bones with a marrow cavity displacing the mineralized cortex distant from neutral bone axis. Vertebral bodies, spring-like shock absorbers on which stiffness is sacrificed for flexibility, show an open-celled porous cancellous structure able to deform and return to its original size and shape without cracking (Seeman, 2003). The structural determinants of bone mechanical strength include width and porosity in the cortical bone; shape, width, connectivity, and anisotropy in the trabecular bone. It has been observed that during age-related bone involution the loss of entire trabeculae would result in higher loads and, consequently, in the thickeing of the remaining elements. In particular, the number of horizontal trabeculae decreases throughout life, whereas vertical trabeculae are resorbed more slowly and tend to increase in width with age (Atkinson, 1967). This is probably due to the fact that the load in vertebral cancellous bone is mainly placed on the vertical elements, thus the compensatory hypertrophy or thickening effect is only expected in the vertical trabeculae. The strength of a vertical trabecula is inversely proportional to the square power of its effective length. This means that a loss of a single horizontal strut or cross-tie increases the effective length of a vertical trabecula by a factor of two, but it will reduce its compressive strength by a factor of four. (Atkinson, 1967). Based on biomechanical characteristics reported above, the evaluation and quantification of these specific properties of bone could provide important information on the fragility status of the skeleton.
From another point of view, it has also been observed (Chesnut III and Rosen, 2001) that the increases in bone mass recorded after drug therapies for osteoporosis can just partly explain the decrease in fracture incidence after these treatments. Therefore, the effect of some of these therapies on microarchitecture could explain their efficacy and rapidity of action. Consequently, there is a growing interest in the quantitative assessment of this particular morphological aspect of bone.
2. Methods for the assessment of microarchitecture The study of microarchitecture is based on the measure of width, number, and separation of trabeculae as well as on their spatial organization. Table 1 shows the list of the main parameters of microarchitecture, which are explained in the next section. The trabecular network, defined as connectivity, is a three-dimensional property that describes the typology of the various connections between the so-called nodes (the structural units that represent the confluence of three or more trabeculae) and the connecting segments (struts and termini). From a quantitative standpoint, connectivity is an index of the variety of links among the trabeculae and can be defined as the major number of connections that can be broken before structure integrity is completely lost (DeHoff et al., 1972). There are several methods to assess bone architecture, particularly at the trabecular level, that is, to detect the organization of bone in space and to measure the complexity of its structure quantitatively. Two different approaches can be identified. (A). The first uses optical microscopy and is based on the principles of histomorphometry (namely, quantitative histology based on stereology), which evaluates microarchitecture on two-dimensional bone sections (Parfitt, 1983). In addition, new stereological methods Table 1 Main histomorphometric parameters of microarchitecture Parameter
Abbreviation
Units
Bone Volume/Tissue Volume (%) Trabecular Number Trabecular Thickness Trabecular Separation Total Skeletal Length Node Number/Tissue Volume Node/Termini Marrow Star Volume Fractal Dimension Trabecular Bone Pattern Factor Interconnectivity Index Euler Number
BV/TV
%
Tb.N Tb.Th Tb.Sp TSL NN/TV N/T MaSV D TBPf
/mm mm mm mm N/mm2 % N/mm3 – –
ICI E/TV
– /mm2
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are now available to provide valuable information on the connectivity of trabecular bone and network complexity (Garrahan et al., 1986). (B). The second applies the most modern diagnostic techniques. Noninvasive physical techniques such as QCT (quantitative computed tomography), especially high resolution computed tomography (hrCT) at 100– 400 mm, volumetric quantitative computed tomography (vQCT) and high resolution magnetic resonance imaging (hrMR) at 100–200 mm have been proposed for the assessment of microarchitecture (Feldkamp et al., 1989; Muller et al., 1998; Laib et al., 2002). The last approaches are interesting and promising but, at this moment, their use is limited because they provide only approximations of micro-structural parameters, with considerable threshold and resolution dependence. Furthermore, some of these approaches are limited by the high exposure to ionizing radiation, and by the expense and limited availability of the equipment. Ultrasound measurements have also been proposed to investigate bone quality, but the poor understanding of what components are measured by broadband attenuation and speed of sound limit their use in this setting (Chappard et al., 1999).
3. Histomorphometric approach The first approaches to quantitative evaluation of trabecular bone structure were based on direct and indirect measure of trabecular width, separation, and number (Wakamatsu and Sisson, 1969; Whitehouse, 1974; Aaron et al., 1987). The measurement of trabecular parameters on the bone sections was initially obtained using a microscope with an ocular equipped with a special grid, such as those shown in Fig. 1. The Trabecular Number is defined as (BV/TV)/Tb.Th, where BV is Bone Volume, TV is the volume of the examined tissue, and Tb.Th is the thickness of the trabecula. The Trabecular Bone Volume (BV/TV) can be also obtained from measurements of bone area (B.Ar)
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and cancellous tissue area (BV/TVZ100!B.Ar/T.Ar). In this approach, Trabecular Thickness, Number, and Separation are derived from trabecular area and perimeter (B.Ar, B.Pm), applying these algorithms: Trabecular Thickness (Tb.ThZ1.199!B.Ar/2/B.Pm; the factor 1.199 is used to correct section obliquity) Trabecular Number (Tb.NZ Tb.Ar!10/Tb.Th, expressed in mm); Trabecular Separation (Tb.Sp: 1000/Tb.N–Tb.Th, expressed in mm) (Chappard et al., 1999). These parameters describe the basic relationship between space and trabecular network. Conventionally, all these parameters are expressed as volume instead of area, even if they are evaluated in twodimensional sections, because they offer an inferred estimation of the spatial organization of the trabecular network. Similarly, the Trabecular Separation, defined as the distance between the edges of the trabeculae, is expressed in three-dimensional units (Parfitt et al., 1987). More recently, new computerized methods that allow the acquisition of more sophisticated measurements by means of a digitizer have been introduced to integrate the use of the microscope (Clermonts and Birkennha¨ger-Frenkel, 1985; Garrahan et al., 1987). These methods supply information on trabecular width as well as on its distribution and on the organization of the different trabeculae in a bone section. In particular, through the process of ‘skeletonization’ of the trabecular bone (that is the measurement of the trabecular profiles and the count of their connections on twodimensional sections by a computed analysis, the so called ‘strut analysis’), it is possible to obtain a network of twodimensional components (the struts), which reproduces the trabecular distribution in space, the so called ‘Trabecular network’ (Garrahan et al., 1986, Fig. 2). The link between three or more struts constitutes a node. When one side of a strut is not joined to a node it is referred to as ‘free end’ or ‘termini’, which represents an interruption in the trabecular network (Parfitt, 1986; Day et al., 2004). The ratio between nodes and termini in a section is an index of spatial connectivity. Other parameters that allow an indirect evaluation of the trabecular connectivity, through the assessment of the marrow connectivity, are:
Fig. 1. The direct measurement of trabecular parameters can be obtained by using an ocular equipped with a special grid. The two panels represent two different grid used for these measurements.
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the mean length of the arrows (three-dimensionally the mean volume) between a random point and a trabecular boundary is higher than the same parameter in the right panel. This value is an evaluation of marrow cavity extension. Consequently, the higher the value, the poorer the trabecular connectivity. Trabecular Bone Pattern Factor (TBPf): is the relation between convex and concave elements (Fig. 4). The rationale of this technique is supported by the fact that in well-connected structure the concave surfaces are abundant, while the convex are more numerous in disconnected structure (Hahn et al., 1992). So, we can consider a concave element as the expression of a wellconnected structure (Chappard et al., 1999; Vogel et al., 1990). In Fig. 4, A is a depiction of a convex figure, while B is a concave structure. When trabecular network is highly disconnected, the convex surfaces (A) are predominant and a dilatation process increases drastically the perimeter, while area is only modestly affected. Opposite trend is observed after a dilatation process of a concave structure. TBPf is defined as: TBPfZ(bone perimeter–bone perimeter after dilatation)/(bone area– bone area after dilatation). Consequently, TBPf is low in well-connected bone and high when highly disconnected trabeculae are observed (Hahn et al., 1992; Chappard et al., 1999). Index of Interconnectivity (ICI): is the connectivity of the marrow cavities which can be evaluated after skeletonization (see above). Terminal ends and the branching nodes of the skeleton are identified and shortest branches are eliminated. Then, the total number of nodes (N), node-to-node branches (NN) and node-to-free
Fig. 2. The strut analysis. The measurement of the trabecular profiles and their connections on two-dimensional sections by a computed analysis (skeletonization) convert the trabecular structure in a network of linear components. The orange points represent the nodes, that is, the connection between three or more struts (segment representing a trabecula, white points). The green points represent the free-ends or termini, which are broken or interrupted elements of trabecular network (For interpretation of the reference to colour in this legend, the reader is referred to the web version of this article).
Marrow Star Volume (MSV): is defined as the mean volume of all the parts of an object when seen unobscured along uninterrupted straight lines in all directions from random points inside the object (Vesterby et al., 1989). In cancellous bone, MSV provides an estimate of the mean size of the marrow space in three-dimensions and thus reflects connectivity. Fig. 3 shows a representation of MSV: in the left panel
A
B
Fig. 3. Schematic drawing to illustrate the concept of the Marrow Star Volume (MSV). This parameter represents the mean volume of all the parts of an object that can be unobscured in all directions from a point inside the object. By the analysis of marrow space distribution, we obtain an indirect evaluation of the trabecular network organization. The higher the mean of the segments the lower the trabecular connectivity. The panel A shows a higher value of MSV (obtained from the mean of the arrow length) than the panel B, highlighting reduced trabecular connectivity.
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A
B
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A
B
C
D
Branches of connection
Fig. 4. Representation of Trabecular Bone Pattern Factor (TBPf). The rationale of TBPf is that in well-connected structure the concave surfaces are abundant, while the convex surfaces are more numerous in disconnected structure. So, we can consider a concave element as the expression of a well-connected structure. When trabecular network is highly disconnected, the convex surfaces are predominant and a dilatation process increases the perimeter of the structure, while area is only modestly affected. The trend is opposite in a well-connected structure. A: Spherical (convex) figureZpoor connected structure; B: figure with concave elementsZwell-connected structure.
end branches (NF) are determined. The number of tree (T) is also obtained (a tree is an independent portion of the medullary space totally enclosed by a trabecular structure). The Index of interconnectivity of the bone marrow is then defined as (N!NN)/[T!(NFC1)]. The higher the interconnectivity of the marrow (characterized by an elevated number of nodes and segmental branches and few trees), the higher the ICI and the fragmentation of the trabecular network (Le et al., 1992). Euler Number (E): expressed per tissue volume (E/TV), i.e. the number of holes minus the number of connected components, which can be interpreted as the maximum number of branches that could be removed without breaking the network into different parts (Feldkamp et al., 1989). Total number of trabecular profiles is indicated with n, while the number of marrow cavities with m. E Z nKm Low value of E indicates a more connected bone. Negative values are obtained from high-connected structure (Chappard et al., 1999). ‘fractal’ analysis or Dimension (D): describes how an object fills space with relation to its structure (Weinsteinm and Majumdar, 1994). The fractal dimension of the trabecular network can be measured by ‘box counting method’ applied on the trabecular image (Fig. 5). A sort of chess board, a grid of boxes, 3, of different sizes is superimposed on the perimeter to be characterized, and the number of boxes that contain the boundary points of the cancellous bone (trabe-
Fig. 5. Representation of Fractal Analysis or Dimension (D). In the boxcounting technique, a grid of boxes, 3, of different sizes is superimposed on the perimeter, and the number of boxes that contain the boundary points of the cancellous bone (trabeculae), N(3), is counted. Based on the graph log N(3) as a function of log 3, the Fractal Dimension (D) can be calculated from the negative slope of the curve. This is an example of the variation in N(3) for a sample (red square) of four different box sizes. A: N(3)Z1; B: N(3)Z4; C: N(3)Z8; D: N(3)Z18 (For interpretation of the reference to colour in this legend, the reader is referred to the web version of this article) .
culae), N(3), are counted. In Fig. 5, an example of the variation in N(3) for a sample (red square) of four different box sizes is shown. The Fractal Dimension (D) is calculated as the negative slope of the linear portion of the curve obtained by plotting the logarithm of N(3) and 3. This parameter represents the smallest number of boxes of side 3 required to cover completely the trabecular boundaries and indicates the perimeter examined with a 3 scale ratio. Each of these parameters gives a distinctive analysis of trabecular bone architecture. This approach is limited by the necessity to infer three-dimensional information from a two-dimensional evaluation. Nevertheless, there are several lines of evidence confirming that measurements on two-dimensional sections are well correlated to the three-dimensional structure and properties of bone (Feldkamp et al., 1989).
4. Microarchitecture and fractures It is well known that exists a direct correlation between Bone Mineral Density (BMD) at the spine and femur and the
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to be a mass-indipendent technique in the evaluation of bone fragility and fracture risk assessment.
risk fracture at these skeletal sites, but it has been also observed a wide overlap in the BMD of patients with and without fractures. As already explained, bone strength is not determined only by mass and BMD. Nevertheless, the combination of many factors can predict more accurately the occurrence of new fractures. With regard to the relationship between microarchitecture and fractures, recent observations seem to confirm that microstructural alterations are important determinants of bone fragility, independently of bone density (Audran et al., 2001; Hordon et al., 2000; Reeker, 1993; Legrand et al., 2000). In the first of these studies (on orchidectomized rats) the evaluation of microarchitectural parameters such as Marrow Star Volume, Fractal Dimension, and node count were dramatically affected. This confirms that bone loss and fragility secondary to orchidectomy was associated with trabecular perforation and reduction of the complexity of trabecular network (Audran et al., 2001). In the other two-dimensional histologic study (Hordon et al., 2000), women with fractures tended to have lower bone surface (BS), wider and fewer trabeculae (Tb.Le and Strut number, respectively), lower mean trabecular plate density (MTPD; known as trabecular number/mm, or Tb.N/mm), fewer trabecular nodes, more termini, wider Trabecular Separation, larger Marrow Star Volume and Trabecular Bone Pattern Factor. In other similar studies significant differences were found in number and thickness of trabecular plates, which were fewer and more widely separated, in Marrow Star Volume (MSV), greater in fracture subject, and number of trabeculae, nodes and free-ends (Hordon et al., 2000; Reeker, 1993). All these data are consistent with loss of connectivity in fracture subjects. Finally, in men affected by idiopathic osteoporosis, Legrand et al. (2000) observed that the differences in bone microarchitecture parameters with the same mass and bone volume were the only discriminating factor between patients with or without vertebral fracture. In conclusion, the histological approach permits us to study accurately bone microarchitecture and demonstrates
A
5. Microarchitecture and treatment Bone microarchitecture seems to be important for understanding the mechanisms of bone fragility as well as the action of the drugs used to prevent osteoporotic fractures. The exact mechanism of the anti-fracture effect of drugs inhibiting resorption has not been completely explained, but increases in bone mass alone cannot account for their efficacy. More attention has lately been paid to the microarchitecture evaluation as a peculiar feature of bone quality, which is one of the targets of the drugs currently in use, such as antiresorptive treatment (bisphosphonates), selective estrogen receptor modulators (SERM, raloxifene) and anabolic agents (teriparatide). For instance, a histomorphometric study on patients undergoing 3-year therapy with alendronate showed that bone lamellar architecture was preserved with no qualitative alteration after treatment (Chavassieux et al., 1997). In a recent study, we found significant differences in terms of microarchitecture in rats treated with glucocorticoids alone or with risedronate (unpublished data, Fig. 6). The administration of risedronate preserved microarchitecture, expressed as direct (NdN/TV, Nd/Tm) and indirect parameters (MSV) with respect to rats treated with glucocorticoids alone. The studies on the use of parathyroid hormone (PTH, teriparatide) also confirm its action on microarchitecture, which can provide an interpretation for its anti-fracture effect from a structural viewpoint (Bradbeer et al., 1992; Dempster et al., 2001). This is even more important if we consider that effects of teriparatide treatment on BMD are controversial. As a matter of fact, after teriparatide a significant increased lumbar BMD and reduced appendicular BMD were observed (Slovik et al., 1986; Hodsman et al., 1997; Lindsay et al., 1997). Nevertheless, few studies have examined the effects of
B
Fig. 6. Different microarchitectural patterns in rats treated with glucorticoids without (A) and with a bisphosphonate (risedronate) (B).
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PTH on bone histology. In a histomorphometric study, biopsy analysis revealed that women treated with teriparatide showed an increased trabecular and cortical thickness, increased BV/TV, with a reduction of MSV (Jang et al., 2003). In these subjects there was evidence of an anabolic effect particularly on the endosteal surface, where an increase in the packet wall width was observed (Dempster et al., 2001). This anabolic effect on cortical bone result to an increased cortical diameter, reduced marrow diameter and, consequently, in a consistent improvement of mechanical strength (Zanchetta et al., 2003). In addition, the evaluation of microarchitecture in rats treated for a long time with PTH showed that its skeletal effects are a complex function of dose and duration. On the other hand, in the rat model, short-term treatment seems to be more effective than near-life treatment, because PTH stimulates skeletal growth throughout life, resulting in abnormal architecture (Sato et al., 2002). Further studies will be necessary to confirm and investigate these acquisitions, in order to understand the exact mechanisms leading to bone fragility and find new and more effective therapeutic strategies to face consequences of osteoporosis and other metabolic bone diseases.
6. Conclusions Microarchitecture is an important element of bone quality and its integrity contributes to the bone mechanical competence. The assessment of microarchitecture may be useful to evaluate the risk of fracture as well as the drug mechanism of action. In the last decade several methods, from the traditional quantitative histology, improved by computed analyses, to the recent applications of computed tomography and magnetic resonance, have been devised and applied to assess this specific bone property. The challenge for the future will be to evaluate bone quality in vivo with the same or better resolution and accuracy than the invasive methods in use today. This goal would be essential to introduce the evaluation of such an important determinant of bone strength as microarchitecture in the routine diagnostic approach to skeletal diseases inducing fragility.
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