Egon Perilli

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.

MicroCT examination of human bone specimens: effects of polymethylmethacrylate embedding on structural parameters

X‐ray microtomography permits the nondestructive investigation of trabecular and cortical bone specimens without special preparation of the sample. To do a quantitative characterization, the cross‐section images have to be binarized, separating bone from nonbone. For this purpose, a widely used method is uniform thresholding. However, for commonly available microtomography scanners which use a polychromatic X‐ray source, it is unclear what effect the surrounding medium (e.g. air, saline solution, polymethylmethacrylate) has on the threshold value used for the binarization. In the literature an easy procedure to find the optimal uniform threshold value for a given acquisition condition is reported. By applying this procedure, the present work investigated whether a microtomography scan of trabecular bone samples in air or embedded in polymethylmethacrylate gave the same results in terms of structural parameters. The gold standard, that is, histological sections, was used as a reference. Two fixed threshold values were found, one for the microtomography scans performed in air and one for the scans with the same samples embedded in polymethylmethacrylate. These were applied on the correspondent microtomography images for the estimation of structural parameters, such as bone volume fraction, direct trabecular thickness, direct trabecular separation and structure model index. Paired comparisons were made in bone volume fraction between histological sections and microtomography cross‐sections for the same bone samples scanned first in air and then embedded in polymethylmethacrylate, by which no significant differences were found. Paired comparisons were also made in bone volume fraction, direct trabecular thickness, direct trabecular separation and structure model index for the same samples over volumes of interest of 4 × 4 × 4 mm3 between microtomography scans in air and scans with the samples embedded in polymethylmethacrylate. Neither these comparisons showed significant differences. This leads to the conclusion that structural parameters estimated by microtomography for human trabecular bone samples scanned either in air or embedded in polymethylmethacrylate are not affected by the surrounding medium (i.e. presence or absence of polymethylmethacrylate), provided that the corresponding optimal threshold value is applied for each acquisition condition.

Application of in vivo micro-computed tomography in the temporal characterisation of subchondral bone architecture in a rat model of low-dose monosodium iodoacetate-induced osteoarthritis.

IntroductionOsteoarthritis (OA) is a complex, multifactorial joint disease affecting both the cartilage and the subchondral bone. Animal models of OA aid in the understanding of the pathogenesis of OA and testing suitable drugs for OA treatment. In this study we characterized the temporal changes in the tibial subchondral bone architecture in a rat model of low-dose monosodium iodoacetate (MIA)-induced OA using in vivo micro-computed tomography (CT).MethodsMale Wistar rats received a single intra-articular injection of low-dose MIA (0.2 mg) in the right knee joint and sterile saline in the left knee joint. The animals were scanned in vivo by micro-CT at two, six, and ten weeks post-injection, analogous to early, intermediate, and advanced stages of OA, to assess architectural changes in the tibial subchondral bone. The articular cartilage changes in the tibiae were assessed macroscopically and histologically at ten weeks post-injection.ResultsInterestingly, tibiae of the MIA-injected knees showed significant bone loss at two weeks, followed by increased trabecular thickness and separation at six and ten weeks. The trabecular number was decreased at all time points compared to control tibiae. The tibial subchondral plate thickness of the MIA-injected knee was increased at two and six weeks and the plate porosity was increased at all time points compared to control. At ten weeks, histology revealed loss of proteoglycans, chondrocyte necrosis, chondrocyte clusters, cartilage fibrillation, and delamination in the MIA-injected tibiae, whereas the control tibiae showed no changes. Micro-CT images and histology showed the presence of subchondral bone sclerosis, cysts, and osteophytes.ConclusionsThese findings demonstrate that the low-dose MIA rat model closely mimics the pathological features of progressive human OA. The low-dose MIA rat model is therefore suitable to study the effect of therapeutic drugs on cartilage and bone in a non-trauma model of OA. In vivo micro-CT is a non-destructive imaging technique that can track structural changes in the tibial subchondral bone in this animal model, and could also be used to track changes in bone in preclinical drug intervention studies for OA treatments.

Failure strength of human vertebrae: Prediction using bone mineral density measured by DXA and bone volume by micro-CT

Significant relationships exist between areal bone mineral density (BMD) derived from dual energy X-ray absorptiometry (DXA) and bone strength. However, the predictive validity of BMD for osteoporotic vertebral fractures remains suboptimal. The diagnostic sensitivity of DXA in the lumbar spine may be improved by assessing BMD from lateral-projection scans, as these might better approximate the objective of measuring the trabecular-rich bone in the vertebral body, compared to the commonly-used posterior-anterior (PA) projections. Nowadays, X-ray micro-computed tomography (μCT) allows non-destructive three-dimensional structural characterization of entire bone segments at high resolution. In this study, human lumbar cadaver spines were examined ex situ by DXA in lateral and PA projections, as well as by μCT, with the aims (1) to investigate the ability of bone quantity measurements obtained by DXA in the lateral projection and in the PA projection, to predict variations in bone quantity measurements obtained by μCT, and (2) to assess their respective capabilities to predict whole vertebral body strength, determined experimentally. Human cadaver spines were scanned by DXA in PA projections and lateral projections. Bone mineral content (BMC) and BMD for L2 and L3 vertebrae were determined. The L2 and L3 vertebrae were then dissected and entirely scanned by μCT. Total bone volume (BV(tot)=cortical+trabecular), trabecular bone volume (BV), and trabecular bone volume fraction (BV/TV) were calculated over the entire vertebrae. The vertebral bodies were then mechanically tested to failure in compression, to determine ultimate load. The variables BV(tot), BV, and BV/TV measured by μCT were better predicted by BMC and BMD measured by lateral-projection DXA, with higher R(2) values and smaller standard errors of the estimate (R(2)=0.65-0.90, SEE=11%-18%), compared to PA-projection DXA (R(2)=0.33-0.53, SEE=22%-34%). The best predictors of ultimate load were BV(tot) and BV assessed by μCT (R(2)=0.88 and R(2)=0.81, respectively), and BMC and BMD from lateral-projection DXA (R(2)=0.82 and R(2)=0.70, respectively). Conversely, BMC and BMD from PA-projection DXA were lower predictors of ultimate load (R(2)=0.49 and R(2)=0.37, respectively). This ex vivo study highlights greater capabilities of lateral-projection DXA to predict variations in vertebral body bone quantity as measured by μCT, and to predict vertebral strength as assessed experimentally, compared to PA-projection DXA. This provides basis for further exploring the clinical application of lateral-projection DXA analysis.

A physical phantom for the calibration of three-dimensional X-ray microtomography examination.

X‐ray microtomography is rapidly gaining importance as a non‐destructive investigation technique, especially in the three‐dimensional examination of trabecular bone. Appropriate quantitative three‐dimensional parameters describing the investigated structure were introduced, such as the model‐independent thickness and the structure model index. The first parameter calculates a volume‐based thickness of the structure in three dimensions independent of an assumed structure type. The second parameter estimates the characteristic form of which the structure is composed, i.e. whether it is more plate‐like, rod‐like or even sphere‐like. These parameters are now experiencing a great diffusion and are rapidly growing in importance. To measure the accuracy of these three‐dimensional parameters, a physical three‐dimensional phantom containing different known geometries and thicknesses, resembling those of the examined structures, is needed. Unfortunately, such particular phantoms are not commonly available and neither does a consolidated standard exist. This work describes the realization of a calibration phantom for three‐dimensional X‐ray microtomography examination and reports an application example using an X‐ray microtomography system. The calibration phantom (external size 13 mm diameter, 23 mm height) was based on various aluminium inserts embedded in a cylinder of polymethylmethacrylate. The inserts had known geometries (wires, foils, meshes and spheres) and thicknesses (ranging from 20 µm to 1 mm). The phantom was successfully applied to an X‐ray microtomography device, providing imaging of the inserted structures and calculation of three‐dimensional parameters such as the model‐independent thickness and the structure model index. With the indications given in the present work it is possible to design a similar phantom in a histology laboratory and to adapt it to the requested applications.

Critical molecular regulators, histomorphometric indices and their correlations in the trabecular bone in primary hip osteoarthritis.

OBJECTIVE This study examined differential gene expression, histomorphometric indices and relationships between these, in femoral trabecular bone from osteoarthritis (OA) patients and control (CTL) subjects, with the aim of identifying potential molecular drivers consistent with changes in structural and remodelling indices in the OA pathology. MATERIALS AND METHODS Bone samples from the intertrochanteric (IT) region were obtained from age and sex-matched cohorts of 23 primary hip OA patients and 21 CTL subjects. Real-time polymerase chain reaction (PCR) and histomorphometric analysis were performed on each sample and correlations between gene expression and histomorphometric variables determined. RESULTS Alterations in gene expression, structural indices and correlations between these were found in OA bone compared to CTL. In OA bone, expression of critical regulators of osteoblast differentiation (TWIST1) and function (PTEN, TIMP4) were decreased, while genes associated with inflammation (SMAD3, CD14) were increased. Bone structural and formation indices (BV/TV, Tb.N, OS/BS) were increased, whereas resorption indices (ES/BS, ES/BV) were decreased. Importantly, significant correlations in CTL bone between CTNNB1 expression and formation indices (OS/BS, OS/BV, OV/BV) were absent in OA bone, indicating altered WNT/β-catenin signalling. TWIST1 expression and BV/TV were correlated in CTL bone, but not in OA bone, consistent with altered osteoblastogenesis in OA. Matrix metalloproteinase 25 (MMP25) expression and remodelling indices (ES/BS, ES/BV, ES/TV) were correlated only in OA pointing to aberrant bone remodelling in this pathology. CONCLUSIONS These findings indicate an altered state of osteoblast differentiation and function in OA driven by several key molecular regulators. In association with this differential gene expression, an altered state of both trabecular bone remodelling and resulting microarchitecture were also observed, further characterising the pathogenesis of primary hip OA.

Micro-CT examination of human bone: from biopsies towards the entire organ

Micro-CT systems are available that facilitate ex vivo examinations of human specimens as big as entire vertebrae, with spatial resolutions in the 10-micrometer range. This opens a new way for looking at entire bones in 3D. Accurate description of the internal microarchitecture of the entire organ can be obtained, at spatial resolutions previously achievable only on excised biopsies. These high resolution scans produce large datasets and come with costs and benefits, which have to be considered in the successful planning of an experiment. The aim of this paper is to present examples of human vertebrae scanned at high resolution (17 µm/pixel), allowing the visualization and quantification of the microarchitecture, and to discuss some aspects of using high resolution scans of such large specimens. The datasets were down-sampled to 34 µm and 68 µm pixel size, and their morphometric parameters compared to those obtained at 17 µm pixel size, in relation to data size and calculation time.

External and internal bone micro-architecture in normal and Kienböck's lunates: A whole-bone micro-computed tomography study

Kienböcks disease is idiopathic osteonecrosis of the lunate, leading to its fracture and collapse. This study compares internal and external bone micro‐architecture of normal and fractured lunates (Kienböcks), by using high‐resolution three‐dimensional (3D) micro‐computed tomography (micro‐CT) on the whole bone of the two lunate types, and histology.

An Innovative CCD-Based High-Resolution CT System for Analysis of Trabecular Bone Tissue

Synchrotron-based digital radiography and microtomography devices are powerful, nondestructive, high-resolution research tools. In this paper, we present a linear system with a pixel size of 22.5 mum and a field-of-view (FOV) 13 cm long and about 1 mm high. The system is composed of a linear converter GOS screen coupled to an intensified electron-bombarded CCD (EBCCD) camera, by means of a rectangular-to-linear fiber optic adapter. This optical guide is composed of seven bundles, each one transporting light in a coherent way to preserve spatial information. In this way, a high spatial resolution over an extended FOV is obtained. The detector works as an X-ray scanner by means of a high-precision translation mechanical device with 18 cm travel range. The total FOV obtained this way is 13 cm long and 18 cm high. The aim of this paper is to demonstrate the feasibility of this system to investigate a large area of a bone and to calculate the appropriate histomorphometric parameters. Here we present an investigation gained at ELETTRA synchrotron facility at Trieste, Italy. A monochromatic 34-keV beam has been used for imaging a human proximal femur, about 9 cm in width, with our system. The reconstructed images (13 cmtimes13 cm) were cross sections containing femoral head, femoral neck, and greater trochanter. The local variations in trabecular and cortical structure of the examined bone were clearly visible at a level not obtainable with medical CT scanners. The used spatial resolution allowed the visualization of thin trabeculae, which typically lie in a range of 100 mum or lower. The quality of the reconstructed cross-section images confirmed that the system presented is a novel tool for high resolution three-dimensional (3-D) imaging of bone structure, with a pixel size over a volume of interest not achievable with conventional microCT scanners

Does cancellous screw insertion torque depend on bone mineral density and/or microarchitecture?

During insertion of a cancellous bone screw, the torque level reaches a plateau, at the engagement of all the screw threads prior to the screw head contact. This plateau torque (T(Plateau)) was found to be a good predictor of the insertion failure torque (stripping) and also exhibited strong positive correlations with areal bone mineral density (aBMD) in ovine bone. However, correlations between T(Plateau) and aBMD, as well as correlations between T(Plateau) and bone microarchitecture, have never been explored in human bone. The aim of this study was to determine whether T(Plateau), a predictor of insertion failure torque, depends on aBMD and/or bone microarchitecture in human femoral heads. Fifty-two excised human femoral heads were obtained. The aBMD and microarchitecture of each specimen were evaluated using dual X-ray Absorptiometry and micro-computed tomography. A cancellous screw was inserted into specimens using an automated micro-mechanical test device, and T(Plateau) was calculated from the insertion profile. T(Plateau) exhibited the strongest correlation with the structure model index (SMI, R=-0.82, p<0.001), followed by bone volume fraction (BV/TV, R=0.80, p<0.01) and aBMD (R=0.76, p<0.01). Stepwise forward regression analysis showed an increase for the prediction of T(Plateau) when aBMD was combined with microarchitectural parameters, i.e., aBMD combined with SMI (R(2) increased from 0.58 to 0.72) and aBMD combined with BV/TV and BS/TV (R(2) increased from 0.58 to 0.74). In conclusion, T(Plateau), a strong predictor for insertion failure torque, is significantly dependent on bone microarchitecture (particularly SMI and BV/TV) and aBMD.