THE 3DIMENSIONAL MICROTOMOGRAPHY ANALYSIS
Variabilità dei parametri morfometrici del tessuto trabecolare umano di campioni con coxo-artrite e osteoporosi
Franco MarinozziI; Andrea MarinozziII; Fabiano BiniI; Francesca ZuppanteI; Raffaella PecciIII; Rossella BediniIII
IDipartimento di Ingegneria Meccanica e Aerospaziale, Sapienza Università di Roma, Rome, Italy
IIUniversità ''Campus Bio-Medico'', Rome, Italy
IIIDipartimento di Tecnologie e Salute, Istituto Superiore di Sanità, Rome, Italy
Morphometric and architectural bone parameters change in diseases such as osteoarthritis and osteoporosis. The mechanical strength of bone is primarily influenced by bone quantity and quality. Bone quality is defined by parameters such as trabecular thickness, trabecular separation, trabecular density and degree of anisotropy that describe the micro-architectural structure of bone. Recently, many studies have validated microtomography as a valuable investigative technique to assess bone morphometry, thanks to micro-CT non-destructive, non-invasive and reliability features, in comparison to traditional techniques such as histology. The aim of this study is the analysis by micro-computed tomography of six specimens, extracted from patients affected by osteoarthritis and osteoporosis, in order to observe the tridimensional structure and calculate several morphometric parameters.
Key words: microtomography, bone morphometry, osteoarthritis, osteoporosis.
I parametri morfometrici e architetturali dell'osso subiscono cambiamenti in presenza di patologie come l'osteoartrite e l'osteoporosi. Il comportamento meccanico dell'osso è influenzato sia dalla quantità (massa ossea) che dalla qualità dell'osso. La qualità dell'osso è definita da parametri che ne caratterizzano la struttura microarchitetturale, tra i principali lo spessore, la separazione e la densità trabecolare e il grado di anisotropia. Recentemente molti studiosi hanno validato la microtomografia come tecnica d'indagine della morfometria ossea e il suo notevole sviluppo è dato dal fatto che a differenza delle tecniche classiche come l'istologia, la microtomografia non provoca il danneggiamento del campione in esame. Nel corrente studio sono stati analizzati sei campioni ossei patologici tramite microtomografia computerizzata, prendendo in esame la struttura tridimensionale e i parametri istomorfometrici.
Parole chiave: microtomografia, morfometria ossea, osteoartrite, osteoporosi.
The morphological and mechanical characterization of trabecular bone has been a widespread research topic in the last years. Trabecular bone consists of a complicated three-dimensional network of plates and rods, arranged in a lattice-like network . Aging, disease process, and therapeutic agents could influence the number of elements in this network, their dimensions and connectivity, thereby causing dramatic changes in bone strength . Until recently, information about structural parameters of trabecular bone were only available by histomorphometry, a destructive procedure limited to two-dimensional analysis. Micro-computed tomography (micro-CT) is an emerging technique for the non- destructive assessment and analysis of the three-dimensional trabecular bone structure. Therefore, it has been largely applied both to basic and pre-clinical bone research throughout the last several years. It relies upon the same physics principles as traditional computed tomography but has a better resolution, down to a few microns. The reconstructed image is created by irradiating the specimen with X-rays and studying the attenuation of the X-rays through the specimen, since the attenuation is directly proportional to the density of the specimen [3-5].
The mechanical strenght of bone is due to bone quantity and micro-architectural structure (quality of bone), therefore, in order to obtain a complete characterization of bone, both characteristics have to be analyzed. Microarchitecture can be assessed by several parameters: trabecular thickness (Tb. Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp). Many studies underlined that these architectural parameters are related to the bone volume fraction (BV/TV) value [6-10].
The evolution of bone mass during life follows height growth. Three age-dependent phases can be distinguished: a bone mass increase until age 20, a consolidation phase, characterized by a continuous and slow increase up to age 35-40, and, finally, a progressive decrease for the rest of life. The loss of a certain amount of bone mass is physiological and unavoidable with age, and is called osteopenia. However, when this process becomes particularly intense and prolonged, enough to induce fractures or trauma, is called osteoporosis (OP).
Osteoporosis is a bone disorder characterized by an inadequate amount and faulty structure of bone, resulting in fractures from relatively minor trauma. It leads to a bone mineral density (BMD) reduction, a bone microarchitecture deterioration and an alteration of the amount and variety of proteins in bone [11, 12].
With aging, other diseases like osteoarthritis may occur. This is a metabolically active process that involves all joint tissues (cartilage, bone, synovium), mainly affecting knees, hips and small hand joints. It is caused by the breakdown and eventual loss of the cartilage of one or more joints. By aging, the water content of the cartilage increases with subsequent decrease in protein content.
In the end, cartilage begins to degenerate by flaking or forming tiny crevasses. In advanced cases, there is a total loss of cartilage cushion between the bones of the joints. Loss of the cartilage cushion causes friction between the bones, leading to pain and limitation of joint mobility can also stimulate new bone outgrowths (osteophytes) to form around the joints [13, 14].
Coxo-arthritis (CA) and osteoporosis are diseases of increasing incidence and prevalence with age [15, 16]. These pathologies and their consequent risk of fracture depend not only on the bone mass but also on the trabecular micro-architecture. The aim of this study is to evaluate some of these aspects on pathological human bone specimens using micro-CT analysis.
Because of their considerable incidence on the population, several studies have analyzed how these diseases affect bone histomorphometric parameters [17, 18].
Ding et al. , by comparing specimens of proximal tibiae from healthy donors and donors affected by osteoarthritis, demonstrated that osteoarthritic specimens show a higher BV/TV and Tb.Th and a lower bone surface to bone volume ratio (BS/BV) than control.
Fazzalari and Parkinson  obtained comparable results by analyzing femoral head specimens from osteoarthritic and healthy donors. Osteoarthritic specimens showed a significant decrease in Tb.N. together with an increase in Tb.Th and Tb.Sp.
Parfitt and Mathews  devised a new method for examining the structural changes that occur in trabecular bone by aging and in osteoporosis. Trabecular bone volume was calculated by measuring two indices, mean trabecular plate thickness (MTPT) and mean trabecular plate density (MTPD). This study confirms that osteoporotic specimens show a reduction in trabecular bone volume that is mainly due to a further reduction in plate density. Similar results were obtained by Chappard and Josselin  comparing twenty-four transiliac bone biopsies from patients with corticosteroid-induced osteoporosis (CSIOP) to control.
Six trabecular bone specimens were extracted from femoral heads of three patients subjected to hip arthroplasty, affected by CA and OP.
Table 1 shows information on collected specimens such as donor sex and a brief description of pathology cases.
After identification, bone specimens were stored at -10 °C for one month then, from the frontal-plane middle-site of the femoral head, a 10 mm-thick slice was obtained for each bone specimen and stored again at -10 °C for 10 hours.
Subsequently, to defat specimens, slices were subjected to three complete cycles of dehydration with aqueous solutions with an increasing percentage of ethanol, 70%, 90% and 99.9%, respectively. Between the dehydration cycles specimens were stored at -10 °C for 10 hours.
Bone specimen slices were then cut with a diamond saw (EXTEC Labcut 1010, Enfield CT) in order to obtain 6 x 6 x 6 mm blocks, that were further dehydrated and defatted by other three cycles with ethanol solutions at different concentrations, as previously described.
Micro-CT acquisition and elaboration
Trabecular bone specimens were acquired using a Skyscan 1072 micro-CT Scanner . The acquisition parameters were set at 100 kV and 98 μA, with 1 mm of aluminum filter and with a voxel size of 11.24 μm for four specimens (CA1, OP1, CA2 and CA4) and 14.66 μm for the other two (OP2 and CA3).
400 projections were acquired over an angular range of 180° by means of steps of 0.45°.
Using the cone beam reconstruction, a software based on the Feldkamp algorithm , projection images in TIFF format and transversal sections of the specimen in BMP format can be obtained.
Analyzing these sets of images with another software, CtAnalyser , the 3D structure and morphometric parameters can be processed. For this purpose, a previous process of binarization is necessary. Binarization needs to choose a threshold value that is the grey level value under which pixel is considered bone and above which pixel is considered as non-bone region. This value was selected by means of an histogram analysis, according to a previous study  that analyzed different methods to choose the appropriate value of threshold.
For each specimen an internal region (ROI) was selected and morphometric parameters for the corresponding volume of interest (VOI) were calculated . The parameters analyzed were defined as follows:
- bone volume/tissue volume (BV/TV, %), an index of bone quantity, being the percentage of the VOI occupied by bone;
- trabecular thickness (Tb.Th, mm) the diameter of the largest sphere which is entirely bounded within the solid surfaces;
- trabecular separation (Tb.Sp, mm) the thickness of the spaces as defined by binarization within the VOI;
- trabecular number (Tb.N, 1/mm) the number of traversals across a trabecular or solid structure made per unit length on a random linear path through the VOI;
-trabecular thickness distribution and trabecular separation distribution obtained by the histogram of Tb.Th and Tb.Sp calculated as a two-pixel interval.
- degree of anisotropy (DA) is a measure of 3D symmetry or the presence or absence of preferential alignment of structures along a particular directional axis and can assume values in the range 0 ÷ 1, where 0 values correspond to a completely isotropic structure and 1 corresponds to a completely anisotropic structure.
Figure 1 shows a characteristic slice for each specimen. A slice represents the transversal sections of the specimen.
The grey level histograms of the specimens obtained by processing these sets of slices are reported in Figure 2A. As described above, in each specimen two representative peaks of bone and air can be observed. Figure 2B shows the common region in which the value of grey level is constant, where analytical threshold was chosen for each specimen. Morphometric parameters and 3D structures of the specimens are shown in Table 2 and Figure 3, respectively.
Differences among specimens can be firstly observed analyzing the reconstructed slices in Figure 1 and their 3D structure in Figure 3, where the trabecular architecture and grey scale representation show the pathology and the withdrawal site.
The processing software allows to calculate not only the average value of Tb.Th and Tb.Sp, as reported in Table 2, but also the distribution of these parameters in the specimen.
Figure 4 and Figure 5 show normalized charts of Tb.Th and Tb.Sp distribution, respectively. These charts present in x-axis the Tb.Th and Tb.Sp value and in y-axis the normalized percentage of trabeculae that has the corresponding thickness and the corresponding separation.
The distributions were fitted by 6th order polynomial interpolation. Correlation coefficients between the data and the fit values are also shown (Table 3). The analysis of these values is fundamental for bone characterization since they allow to obtain an accurate estimation of bone architecture.
Moreover, these parameters undergo considerable changes in bone diseases. In particular, diseases such as osteoporosis, cause a decrease of the Tb.N and Tb.Th, and conversely an increase of the Tb.Sp value .
Since it is known that a denser material has a higher attenuation coefficient of X-rays, a grey level of an image obtained by an X-ray irradiation of a specimen can be correlated with density of the material so the grey level of each voxel represents the mineral content of the trabecular bone . According to this and observing the histograms reported in Figure 2A, it can be observed that a peaky distribution, which could be seen for the OP specimens OP1 and OP2 and CA specimens CA3 and CA4 indicates a high trabecular tissue density. Conversely, CA1 and CA2 specimens exhibit a broader distribution around the highest grey levels and thus a lower density of the trabecular tissue.
This can also be observed in Figure 1, where slices of specimens characterized by a peaky distribution of grey levels present a higher difference between trabecular structure and marrow canals.
Trabecular thickness and separation
Table 2 shows that CA1 specimen has the thinnest Tb.Th population with a relatively narrow peak of 0.15 mm, CA1 also shows a relatively broad distribution with a peak value of Tb.Sp of 0.62 mm (Figure 5). The Tb.N is 1.08 per millimeter, that is one of the lower value among all analyzed specimens. These morphometric parameters can explain the low value of BV/TV as compared to the other specimens.
A similar performance has been obtained as regards OP1.
OP1 specimen shows the highest value of Tb.Th and Tb.Sp, and a very broad distribution of Tb.Sp (Figure 5).
A broad distribution of Tb.Th and Tb.Sp is also observed in OP2, that shows the highest value of BV/TV and also of Tb.Th (Figure 1).
CA2 features a Tb.Sp rather broad distribution with two peaks.
Finally, CA3 and CA4 are characterized by similar values, and also peak distributions, of Tb.Th and Tb.Sp.
CA3 and CA4 show the highest value of Tb.N .and their high trabecular density can also be observed in Figure 1.
Evaluating all specimens, DA is in the range of 0.2-0.7, being CA2 the most isotropic specimen and OP1 the most anisotropic one, respectively.
This study shows that micro-CT is a promising technique for trabecular bone analysis. Bone morphometric parameters obtained by microtomographic processing allows to completely characterize human bone.
This analysis is also useful to compare human bone specimens with different pathologies in order to evaluate a qualitative and quantitative link between various pathologies by means of ultrastructural images and morphometric parameters.
By comparing morphometric values shown in Table 2 with the respective gender and pathologies shown in Table 1, it is clear that microtomographic analysis results are in agreement with clinical data and data obtained using traditional analysis techniques .
The anisotropy of trabecular bone differs according to skeletal sites, and, as underlined by anatomical studies, trabecular bone femoral head is one of the highest anisotropic sites. Trabecular bone is an anisotropic material but the highest anisotropy value found in osteoporosis specimens is in accordance with the findings of Newitt et al.  who reported that the increase of bone resorption in osteoporosis leads to an increase of anisotropy.
Interestingly, in this study, Tb.Sp has obtained the highest values in the two samples of osteoporotic bone, as one might expect.
The correlation between the data obtained and the fit value has been performed in Table 3. High values of both parameters (Tb.Th and Tb.Sp) are evidence of the adequacy of the data obtained, with the only exception of one osteoporotic specimen.
An analysis of this kind can be helpful in in vitro bone studies, through diagnostic techniques, and may contribute to drug development for treatment of pathologies considered in this study.
However, to validate the promising results of this study, a considerable number of specimens should be collected and analyzed for each pathology, to gain statistical significance.
Moreover, a reliable threshold technique should be developed and applied, in order to reduce the uncertainty by which the morphometric parameters are processed.
Conflict of interest statement
There are no potential conflicts of interest or any financial or personal relationships with other people or organizations that could inappropriately bias conduct and findings of this study.
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▲ Address for correspondence:
Dipartimento di Ingegneria Meccanica e Aerospaziale, Sapienza Università di Roma
Via Eudossiana 18
00184 Rome, Italy
Submitted on invitation.
Accepted on 19 December 2011.