3D histomorphometric quantification of trabecular bones by computed microtomography using synchrotron radiation

Conventional bone histomorphometry is an important method for quantitative evaluation of bone microstructure. X-ray computed microtomography is a non-invasive technique, which can be used to evaluate histomorphometric indices in trabecular bones (BV/TV, BS/BV, Tb.N, Tb.Th, Tb.Sp). In this technique, 3D images are used to quantify the whole sample, differently from the conventional one, in which the quantification is performed in 2D slices and extrapolated for 3D case. In this work, histomorphometric quantification using synchrotron 3D X-ray computed microtomography was performed to quantify the bone structure at different skeletal sites as well as to investigate the effects of bone diseases on quantitative understanding of bone architecture. The images were obtained at Synchrotron Radiation for MEdical Physics (SYRMEP) beamline, at ELETTRA synchrotron radiation facility, Italy. Concerning the obtained results for normal and pathological bones from same skeletal sites and individuals, from our results, a certain declining bone volume fraction was achieved. The results obtained could be used in forming the basis for comparison of the bone microarchitecture and can be a valuable tool for predicting bone fragility.     

X射线显微CT用于大鼠骨小梁结构分析的研究

应用X射线显微CT(X-μCT)对正常及骨质疏松大鼠的骨小梁结构进行了分析,并与骨组织形态计量法的测量值进行了比较,探讨了X射线光谱技术在骨结构分析中的应用。实验对大鼠骨样品进行X-μCT扫描,扫描条件为 80 kVp,80 μA,360°旋转,帧平均4帧,角度增益 0.4°,分辨率14 μm。三维重建并分析了骨小梁结构,结构参数包括骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数量(Tb.N)以及骨小梁间隔(Tb.Sp)。结果表明,采用X-μCT分析不同组大鼠的骨小梁结构参数值之间存在显著差异(P<0.05),测定值与传统骨组织形态计量法的测定值显著相关,其中胫骨骨小梁BV/TV,Tb.Th,Tb.N,Tb.Sp的相关系数r分别为0.984,0.960,0.995,0.988,腰椎骨小梁各结构参数的相关系数分别为0.938,0.968,0.877,0.951。因此,X-μCT可以较好地呈现并区分正常骨组织、骨质疏松骨组织以及经雌激素治疗后骨组织的微观结构,可以实现对骨小梁结构参数的分析测定,与骨组织形态计量法相比是一种更精确、立体、快速且无损测量骨微结构和评价骨质量的方法。     

Unwrapping microcomputed tomographic images for measuring cortical osteolytic lesions in the 5T2 murine model of myeloma treated by bisphosphonate

Multiple myeloma is due to the proliferation of malignant plasma cells which increase the number of osteoclasts leading to trabecular and cortical bone osteolysis. The 5T2MM murine model reproduces the human disease and microcomputed tomography is a precise tool to investigate bone loss. Bisphosphonates (zoledronic acid or pamidronate) are used in preventing osteolysis. However, loss of cortical bone in not possible to quantify by histomorphometry on histological sections or microCT images.Osteolysis was studied in mice grafted with the 5THL subline to see if one drug was more active after 10 weeks. Mice were distributed into 4 groups: control, untreated, treated with pamidronate or with zoledronic acid. The left femurs were embedded undecalcified and sectioned at 7 μm. The right tibias and femurs were analyzed by microCT and trabecular morphometric parameters were obtained. Cortical bone osteolysis was analyzed by developing a new algorithm to unwrap microCT sections of the cortices, allowing measurement of the number of perforations, porosity and mean perforation area.The bisphosphonates had no significant effect on the tumor growth as evidence by the absence of effect on the M-protein level. Cortical perforations were evidenced on histological sections and their number seemed to be reduced by both bisphosphonates. MicroCT was used to quantify the trabecular bone: a bone loss was evidenced in the untreated myeloma group and both bisphosphonates appeared equal to preserve trabecular mass. However, the number and size of cortical perforations cannot be determined on 3D models. Unwrapping microCT images provided flat images allowing a precise determination of cortical perforations. Pamidronate did not reduce the number and size of cortical perforations but significantly reduced porosity. Zoledronic acid appeared significantly superior and considerably reduced all parameters.Unwrapping microCT image is a new method allowing the measurement of cortical perforations in bone malignancies, a parameter that cannot be measured correctly on 2D histological sections.     

Reproducibility of the quantitative assessment of cartilage morphology and trabecular bone structure with magnetic resonance imaging at 7 T

To assess the reproducibility of quantitative measurements of cartilage morphology and trabecular bone structure of the knee at 7 T, high-resolution sagittal spoiled gradient-echo images and high-resolution axial fully refocused steady-state free-precession (SSFP) images from six healthy volunteers were acquired with a 7-T scanner. The subjects were repositioned between repeated scans to test the reproducibility of the measurements. The reproducibility of each measurement was evaluated using the coefficient(s) of variation (CV). The computed CV were 1.13% and 1.55% for cartilage thickness and cartilage volume, respectively, and were 2.86%, 1.07%, 2.27% and 3.30% for apparent bone volume over total volume fraction (app.BV/TV), apparent trabecular number (app.Tb.N), apparent trabecular separation (app.Tb.Sp) and apparent trabecular thickness (app.Tb.Th), respectively. The results demonstrate that quantitative assessment of cartilage morphology and trabecular bone structure is reproducible at 7 T and motivates future musculoskeletal applications seeking the high-field strength's superior signal-to-noise ratio.     

Interrelationships between 3-T-MRI-derived cortical and trabecular bone structure parameters and quantitative-computed-tomography-derivedbone mineral density

Recently, 3-T magnetic resonance imaging (MRI) has been introduced for bone imaging. Through higher signal-to-noise ratios, as compared to 1.5-T MRI, it promises to be a more powerful tool for the assessment of cortical and trabecular bone measures. The goal of our study was to compare MRI-derived cortical and trabecular bone measures to quantitative computed tomography (QCT)-derived bone mineral density (BMD). Using 3-T MRI in 51 postmenopausal women, apparent (app.) measures of bone volume/total volume, trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation were derived at the distal radius, distal tibia and calcaneus. Cortical thickness (Ct.Th) was calculated at the distal radius and distal tibia. These measures were compared to QCT-derived BMD of the spine, hip and radius. Significant correlations (^?P<.05; ^??P<.001; ^???P<.0001) were found between spine BMD- and MRI-derived Ct.Th (r_radius=.55, ^?P<.05; r_tibia=.67, ^???P<.0001) and app. Tb.N (r_radius=.33, ^?P<.05; r_tibia=.35, ^?P<.05) at the radius and tibia. Furthermore, within the first 10 mm at the radius, an inverse correlation for Ct.Th and app. BV/TV (r_6mm=−.56, P<.001; r_10mm=−.36, P<.05) and app. Tb.Th (r_6mm=−.54, P<.001; r_10mm=−.41, P<.05) was found.     

Wavelet-based characterization of vertebral trabecular bone structure from magnetic resonance images at 3 T compared with micro-computed tomographic measurements

Trabecular bone structure and bone density contribute to the strength of bone and are important in the study of osteoporosis. Wavelets are a powerful tool in characterizing and quantifying texture in an image. The purpose of this study was to validate wavelets as a tool in computing trabecular bone thickness directly from gray-level images. To this end, eight cylindrical cores of vertebral trabecular bone were imaged using 3-T magnetic resonance imaging (MRI) and micro-computed tomography (microCT). Thickness measurements of the trabecular bone from the wavelet-based analysis were compared with standard 2D structural parameters analogous to bone histomorphometry (MR images) and direct 3D distance transformation methods (microCT images). Additionally, bone volume fraction was determined using each method. The average difference in trabecular thickness between the wavelet and standard methods was less than the size of 1 pixel size for both MRI and microCT analysis. A correlation (R) of .94 for microCT measurements and that of .52 for MRI were found for the bone volume fraction. Based on these results, we conclude that wavelet-based methods deliver results comparable with those from established MR histomorphometric measurements. Because the wavelet transform is more robust with respect to image noise and operates directly on gray-level images, it could be a powerful tool for computing structural bone parameters from MR images acquired using high resolution and thus limited signal scenarios.     

X-ray imaging characterization of femoral bones in aging mice with osteopetrotic disorder

Aging mice with a rare osteopetrotic disorder in which the entire space of femoral bones are filled with trabecular bones are used as our research platform. A complete study is conducted with a micro computed tomography (CT) system to characterize the bone abnormality. Technical assessment of femoral bones includes geometric structure, biomechanical strength, bone mineral density (BMD), and bone mineral content (BMC). Normal aging mice of similar ages are included for comparisons. In our imaging work, we model the trabecular bone as a cylindrical rod and new quantitative which are not previously discussed are developed for advanced analysis, including trabecular segment length, trabecular segment radius, connecting node number, and distribution of trabecular segment radius. We then identified a geometric characteristic in which there are local maximums (0.0049, 0.0119, and 0.0147 mm) in the structure of trabecular segment radius. Our calculations show 343% higher in percent trabecular bone volume at distal-metaphysis; 38% higher in cortical thickness at mid-diaphysis; 11% higher in cortical cross-sectional moment of inertia at mid-diaphysis; 42% higher in cortical thickness at femur neck; 26% higher in cortical cross-sectional moment of inertia at femur neck; 31% and 395% higher in trabecular BMD and BMC at distal-metaphysis; 17% and 27% higher in cortical BMD and BMC at distal-metaphysis; 9% and 53% higher in cortical BMD and BMC at mid-diaphysis; 25% and 64% higher in cortical BMD and BMC at femur neck. Our new quantitative parameters and findings may be extended to evaluate the treatment response for other similar bone disorders.     

Surface bioactivity modification of titanium by CO2 plasma treatment and induction of hydroxyapatite: In vitro and in vivo studies

Since metallic biomaterials used for orthopedic and dental implants possess a paucity of reactive functional groups, bioactivity modification of these materials is challenging. In the present work, the titanium discs and rods were treated with carbon dioxide plasma and then incubated in a modified simulated body fluid 1.5SBF to obtain a hydroxyapatite layer. Surface hydrophilicity of samples, changes of surface chemistry, surface morphologies of samples, and structural analysis of formed hydroxyapatite were investigated by contact angle to water, X-ray photoelectron spectrometer (XPS), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and X-ray diffraction (XRD). The results demonstrated that hydrophilicity of titanium surface was improved and hydroxyl groups increased after modification with carbon dioxide plasma treatment. The hydroxyl groups on the surface of titanium were the richest after carbon dioxide plasma treatment under the condition of 20 W for less than 30 s. The hydroxyapatite formability of titanium surface was enhanced by carbon dioxide plasma pretreatment, which was attributed to the surface chemistry. MC3T3-E1 cell as a model cell was cultured on the Ti, CPT-Ti and CPT/SBF-Ti discs in vitro, and the results of the morphology and differentiation of the cell showed that CPT/SBF-Ti was the highest bioactive. The relative parameters of the new bone around the Ti and CPT/SBF-Ti rods including bone mineral density (BMD), a ratio of bone volume to total volume (BV/TV), trabecular thickness (Tb.Th.) and trabecular number (Tb.N.) were analyzed by a micro-computed tomography (micro-CT) after 4-, 8- and 12-week implantation periods in vivo. The results indicated that the CPT/SBF-Ti was more advantageous for new bone formation.     

Short-TE projection reconstruction NMR microscopy of trabecular bone.

The aim of this study was to assess the potential of projection reconstruction (PR) NMR microscopy in the quantitative evaluation of trabecular bone architecture. Short-TE PR spin-echo microimages were acquired at 7.05 T on normal bone explants. The main structural parameters such as bone volume fraction (BVF), trabecular thickness (Tb.Th.) and trabecular separation (Tb.Sp.) were obtained from the 3D microimages using the method of directed secants. Quantitative structural data were then compared with those derived from conventional spin-echo microimages. Our study indicates that projection reconstruction NMR microscopy promises to be more accurate than the conventional FTI method in the analysis of trabecular bone.     

Fast wave ultrasonic propagation in trabecular bone: numerical study of the influence of porosity and structural anisotropy

Our goal is to assess the potential of computational methods as an alternative to analytical models to predict the two longitudinal wave modes observed in cancellous bone and predicted by the Biot theory. A three-dimensional (3D) finite-difference time-domain method is coupled with 34 human femoral trabecular microstructures measured using microcomputed tomography. The main trabecular alignment (MTA) and the degree of anisotropy (DA) were assessed for all samples. DA values were comprised between 1.02 and 1.9. The influence of bone volume fraction (BV/TV) between 5% and 25% on the properties of the fast and slow waves was studied using a dedicated image processing algorithm to modify the initial 3D microstructures. A heuristic method was devised to determine when both wave modes are time separated. The simulations (performed in three perpendicular directions) predicted that both waves generally overlap in time for a direction of propagation perpendicular to the MTA. When these directions are parallel, both waves are separated in time for samples with high DA and BV/TV values. A relationship was found between the least bone volume fraction required for the observation of nonoverlapping waves and the degree of anisotropy: The higher the DA, the lower the least BV/TV.     

A method for vibrational assessment of cortical bone

Large bones from many anatomical locations of the human skeleton consist of an outer shaft (cortex) surrounding a highly porous internal region (trabecular bone) whose structure is reminiscent of a disordered cubic network. Age related degradation of cortical and trabecular bone takes different forms. Trabecular bone weakens primarily by loss of connectivity of the porous network, and recent studies have shown that vibrational response can be used to obtain reliable estimates for loss of its strength. In contrast, cortical bone degrades via the accumulation of long fractures and changes in the level of mineralization of the bone tissue. In this paper, we model cortical bone by an initially solid specimen with uniform density to which long fractures are introduced; we find that, as in the case of trabecular bone, vibrational assessment provides more reliable estimates of residual strength in cortical bone than is possible using measurements of density or porosity.     

Attenuation in trabecular bone: A comparison between numerical simulation and experimental results in human femur

Numerical simulations (finite-difference time domain) are compared to experimental results of ultrasound wave propagation through human trabecular bones. Three-dimensional high-resolution microcomputed tomography reconstructions served as input geometry for the simulation. The numerical simulation took into account scattering, but not absorption. Simulated and experimental values of the attenuation coefficients (alpha, dB/cm) and the normalized broadband ultrasound attenuation (nBUA, dB/cm/MHz) were measured and compared on a set of 28 samples. While experimental and simulated nBUA values were highly correlated (R(2)=0.83), and showed a similar dependence with bone volume fraction, the simulation correctly predicted experimental nBUA values only for low bone volume fraction (BV/TV). Attenuation coefficients were underestimated by the simulation. The absolute difference between experimental and simulated alpha values increased with both BV/TV and frequency. As a function of frequency, the relative difference between experimental and simulated alpha values decreased from 60% around 400 kHz to 30% around 1.2 MHz. Under the assumption that the observed discrepancy expresses the effect of the absorption, our results suggests that nBUA and its dependence on BV/TV can be mostly explained by scattering, and that the relative contribution of scattering to alpha increases with frequency, becoming predominant (>50 %) over absorption for frequencies above 600 kHz.     

Ultrasonic wave propagation in cancellous and cortical bone: prediction of some experimental results by Biot's theory.

Pulse transmission ultrasound was used to determine the longitudinal wave speed along the direction of trabecular alignment in 32 water-saturated anisotropic tibial bovine cancellous bone samples and in one cortical bone sample also from the bovine tibia. These results are compared to published ultrasound wave-speed data obtained from bovine femoral specimens. Nonlinear regression was used to fit Biot's theory to the data. The correlation coefficient for regression analysis between the experimental ultrasound velocities and the velocities predicted by Biot's theory was r = 0.78.     

Influence of the precision of spectral backscatter measurements on the estimation of scatterers size in cancellous bone

The goal of this study is to propose a model for the ultrasonic frequency-dependent backscatter coefficient in femoral cancellous bone. This model has been developed with success to predict backscatter in human calcaneal bone [Jenson, Ultr. Med. Biol. 2003]. A weak scattering model is used and the backscatter coefficient is expressed in terms of a Gaussian autocorrelation function of the medium. The backscatter coefficient is computed and comparison is made with experimental data for 37 specimens and for frequency ranging from 0.4 to 1.2 MHz. An excellent agreement between experimental data and predictions is found for both the magnitude and the frequency-dependence of the backscatter coefficient. Then, a nonlinear regression is performed for each specimen, and the mean trabecular thickness is estimated. Experimental data and theoretical predictions are averaged over the 37 specimens. We also find a close agreement between theoretical predictions obtained using the Gaussian autocorrelation function (scatterer size=134+/-15 microm) and the mean trabecular thickness (Tb.Th=132+/-12 microm) derived from the analysis of bone 3-D micro-architecture using high-resolution micro-tomography. However, the correlation between individual experimental and estimated Tb.Th values is moderate (R(2)=0.44). The performance of the estimator are limited mainly by two factors: interference noise due to random positioning of the scatterers and attenuation. We show that the fundamental limitation of our estimator due to the speckle noise is around 5 microm for trabecular thickness estimation. This limitation is lower than the observed biological variability which is around 30 microm and should not be a limiting factor for individual prediction. A second limitation is the tremendous attenuation encountered in highly scattering media such as cancellous bone, which results in highly damped backscatter signals. The compensation for attenuation is difficult to perform, and it may be a critical point that limits the precision of the estimator.     

Effects of swimming associated with risedronate in osteopenic bones: An experimental study with ovariectomized rats

Physical activity and risedronate sodium have effects on metabolic bone diseases, maintaining the integrity of bone tissue. Our objective was to evaluate the effects of swimming associated with risedronate as a prophylactic means in osteopenic bone of ovariectomized rats. A total of 24 animals of the Wistar strain were used and separated into four groups containing six animals: Ovariectomy (OVX), ovariectomy and swimming (OVXS), ovariectomy and risedronate (OVXM), ovariectomy, risedronate and swimming (OVXMS). The effectiveness of the treatments were evaluated using the tibia by means of biomechanical, radiographic, histomorphometric analyzes. Statistical analysis was performed by the non-parametric Kruskal–Wallis test (p < 0.05). The OVXM and OVXMS groups showed higher values ​​compared to OVX in maximum strength and rigidity. Microscopic analysis showed increased trabecular bone in the OVXM group in relation to the others, and in the OVXMS compared to OVXS. Proximal densitometry in the OVXM and OVXMS groups showed higher values than the OVX and OVXS groups. There were no significant differences in overall densitometry. In conclusion, when comparing the prophylactic means, risedronate was able to preserve bone mass significantly, unlike exercise where an improvement of bone tissue was observed, although not significant, and when swimming and risedronate are combined the result was even better.     

Influence of cancellous bone microstructure on two ultrasonic wave propagations in bovine femur: an in vitro study

The influence of cancellous bone microstructure on the ultrasonic wave propagation of fast and slow waves was experimentally investigated. Four spherical cancellous bone specimens extracted from two bovine femora were prepared for the estimation of acoustical and structural anisotropies of cancellous bone. In vitro measurements were performed using a PVDF transducer (excited by a single sinusoidal wave at 1 MHz) by rotating the spherical specimens. In addition, the mean intercept length (MIL) and bone volume fraction (BV/TV) were estimated by X-ray micro-computed tomography. Separation of the fast and slow waves was clearly observed in two specimens. The fast wave speed was strongly dependent on the wave propagation direction, with the maximum speed along the main trabecular direction. The fast wave speed increased with the MIL. The slow wave speed, however, was almost constant. The fast wave speeds were statistically higher, and their amplitudes were statistically lower in the case of wave separation than in that of wave overlap.     

Dependence of threshold variations on the assessment of histomorphometric indices from computed microtomography using synchrotron radiation

In this work, the variation of threshold level for segmentation of tomographic image in a micron scale accomplished with synchrotron radiation was studied. Three different sites of rat femora were evaluated from computed microtomography performed at SYRMEP beamline of ELETTRA Synchrotron Laboratory with a resolution of 9 μm. An optimal threshold level was selected and then varied in two points up and down in steps of one point, corresponding to 0.39% per point. Histomorphometric indices were calculated directly from the three dimensional segmented images of trabecular microstructure. The results showed that histomorphometric indices presented small variations for all indices in all femur sites. The greater variation occurred for trabecular thickness (Tb.Th) [from ?2.84% to 2.99%] while the smaller one occurred for trabecular number (Tb.N) [?0.26% to 0.35].     

The TEM characterization of the lamellar structure of osteoporotic human trabecular bone

The lamellar structure of osteoporotic human trabecular bone was characterized experimentally by means of transmission electron microscopy (TEM). More specifically, the TEM was used to determine if trabecular bone exhibits similar lamellar structural motifs as cortical bone by analyzing unmineralized, mineralized and demineralized bone, and to study the influence of the osteocyte network on the lamellar structure of osteoporotic trabecular bone. Comparison with normal trabecular bone is included. This paper summarizes partial results of a larger study, which addressed the characterization of the hierarchical structure of normal versus osteoporotic human trabecular bone [Rubin, M.A., 2001. Multiscale characterization of the ultrastructure of trabecular bone in osteoporotic and normal humans and in two inbred strains of mice. MS Thesis, Georgia Institute of Technology.] at several structural scales.     

Influence of low-intensity pulsed ultrasound on osteogenic tissue regeneration in a periodontal injury model: X-ray image alterations assessed by micro-computed tomography

Objective

This study was conducted to evaluate, with micro-computed tomography, the influence of low-intensity pulsed ultrasound on wound-healing in periodontal tissues.

Methods

Periodontal disease with Class II furcation involvement was surgically produced at the bilateral mandibular premolars in 8 adult male beagle dogs. Twenty-four teeth were randomly assigned among 4 groups (G): G1, periodontal flap surgery; G2, periodontal flap surgery + low-intensity pulsed ultrasound (LIPUS); G3, guided tissue regeneration (GTR) surgery; G4, GTR surgery plus LIPUS. The affected area in the experimental group was exposed to LIPUS. At 6 and 8 weeks, the X-ray images of regenerated teeth were referred to micro-CT scanning for 3-D measurement.

Results

Bone volume (BV), bone surface (BS), and number of trabeculae (Tb) in G2 and G4 were higher than in G1 and G3 (p < 0.05). BV, BS, and Tb.N of the GTR + LIPUS group were higher than in the GTR group. BV, BS, and Tb.N of the LIPUS group were higher than in the periodontal flap surgery group.

Conclusion

LIPUS irradiation increased the number, volume, and area of new alveolar bone trabeculae. LIPUS has the potential to promote the repair of periodontal tissue, and may work effectively if combined with GTR.     

Infrared optical imaging of matrix metalloproteinases (MMPs) up regulation following ischemia reperfusion is ameliorated by hypothermia

Background

Continuous theta burst stimulation (cTBS) is a form of repetitive transcranial magnetic stimulation which has been shown to alter cortical excitability in the upper limb representation of primary somatosensory cortex (SI). However, it is unknown whether cTBS modulates cortical excitability within the lower limb representation in SI. The present study investigates the effects of cTBS over the SI lower limb representation on cortical somatosensory evoked potentials (SEPs) and Hoffmann reflex (H-reflex) following tibial nerve stimulation at the knee. SEPs and H-reflex were recorded before and in four time blocks up to 30 minutes following cTBS targeting the lower limb representation within SI.

Results

Following cTBS, the P1-N1 first cortical potential was significantly decreased at 12?C16 minutes. CTBS also suppressed the P2-N2 second cortical potential for up to 30 minutes following stimulation. The H-reflex remained statistically unchanged following cTBS although there was a modest suppression observed.

Conclusion

We conclude that cTBS decreases cortical excitability of the lower limb representation of SI as evidenced by suppressed SEP amplitude. The duration and magnitude of the cTBS after effects are similar to those observed in upper limb studies.