Ara Nazarian

Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson–Gilford progeria syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is an extremely rare, fatal, segmental premature aging syndrome caused by a mutation in LMNA that produces the farnesylated aberrant lamin A protein, progerin. This multisystem disorder causes failure to thrive and accelerated atherosclerosis leading to early death. Farnesyltransferase inhibitors have ameliorated disease phenotypes in preclinical studies. Twenty-five patients with HGPS received the farnesyltransferase inhibitor lonafarnib for a minimum of 2 y. Primary outcome success was predefined as a 50% increase over pretherapy in estimated annual rate of weight gain, or change from pretherapy weight loss to statistically significant on-study weight gain. Nine patients experienced a ≥50% increase, six experienced a ≥50% decrease, and 10 remained stable with respect to rate of weight gain. Secondary outcomes included decreases in arterial pulse wave velocity and carotid artery echodensity and increases in skeletal rigidity and sensorineural hearing within patient subgroups. All patients improved in one or more of these outcomes. Results from this clinical treatment trial for children with HGPS provide preliminary evidence that lonafarnib may improve vascular stiffness, bone structure, and audiological status.

Time-lapsed microstructural imaging of bone failure behavior

Many bones within the axial and appendicular skeleton are subjected to repetitive loading during the course of ordinary daily activities. If this loading is of sufficient magnitude or duration, failure of the bone tissue may result. Until recently the structural analysis of these fractures has been limited to two-dimensional sections. Due to the inherent destructiveness of this method, dynamic assessment of fracture progression has not been possible. An image-guided technique to analyze structural failure has been developed utilizing step-wise micro-compression in combination with time-lapsed micro-computed tomographic imaging. This technique allows, for the first time, direct three-dimensional visualization and quantification of fracture initiation and progression on the microscopic level and relates the global failure properties of trabecular bone to those of the individual trabeculae. The goals of this project were first to design and fabricate a novel micro-mechanical testing system, composed of a micro-compression device and a material testing and data acquisition system; and second, to validate the testing system to perform step-wise testing of trabecular bone specimens based on image-guided failure analysis. Due to the rate dependant properties of bone, stress relaxation was a concerning factor with respect to the step-wise testing method. In order to address these concerns, the results of the step-wise testing method were compared to those obtained from a conventional continuous test (considered to be the gold standard for the step-wise compressive mechanical testing) over the same total strain range and testing conditions. This was performed using porous aluminum alloy samples with highly reproducible and homogenous structural properties as well as trabecular bone samples from a single whale vertebra. Five cylinders from aluminum foam and trabecular whale bone each were compressed and imaged in a step-wise fashion from 0% to 20% strain at intervals of 2%, 4%, 8%, 12%, 16% and 20%. Mechanical properties obtained from the continuous and step-wise methods were not significantly different for both aluminum foam and whale bone specimens (p>0.05). Both testing methods yielded very similar stress-strain graphs with almost identical elastic and plastic regions with overlaying standard error bars for both whale bone and aluminum foam specimens. This was further concurred by performing regression analyses between the stress data from both testing methods (r(2)=0.98 for whale bone and aluminum foam specimens). Animations of fracture initiation and progression revealed that failure always occurred in local bands with the remaining regions of the structure largely unaffected independent of structure type. In conclusion, we found step-wise micro-compression to be a valid approach for image-guided failure assessment (IGFA) with high precision and accuracy as compared to classical continuous testing. We expect findings from upcoming studies of IGFA of human vertebral bone to improve our understanding of the relative importance of densitometric, morphological, and loading factors in the etiology of spontaneous fractures of the spine. Eventually, this improved understanding may lead to more successful approaches to the prevention of age-related fatigue fractures.

Direct Percutaneous Gene Delivery to Enhance Healing of Segmental Bone Defects

BACKGROUND Healing of segmental bone defects can be induced experimentally with genetically modified osteoprogenitor cells, an ex vivo strategy that requires two operative interventions and substantial cost. Direct transfer of osteogenic genes offers an alternative, clinically expeditious, cost-effective approach. We evaluated its potential in a well-established, critical-size, rat femoral defect model. METHODS A critical-size defect was created in the right femur of forty-eight skeletally mature Sprague-Dawley rats. After twenty-four hours, each defect received a single, intralesional, percutaneous injection of adenovirus carrying bone morphogenetic protein-2 (Ad.BMP-2) or luciferase cDNA (Ad.luc) or it remained untreated. Healing was monitored with weekly radiographs. At eight weeks, the rats were killed and the femora were evaluated with dual-energy x-ray absorptiometry, micro-computed tomography, histological analysis, histomorphometry, and torsional mechanical testing. RESULTS Radiographically, 75% of the Ad.BMP-2-treated femora showed osseous union. Bone mineral content was similar between the Ad.BMP-2-treated femora (0.045 +/- 0.020 g) and the contralateral, intact femora (0.047 +/- 0.003 g). Histologically, 50% of the Ad.BMP-2-treated defects were bridged by lamellar, trabecular bone; the other 50% contained islands of cartilage. The control (Ad.luc-treated) defects were filled with fibrous tissue. Histomorphometry demonstrated a large difference in osteogenesis between the Ad.BMP-2 group (mean bone area, 3.25 +/- 0.67 mm(2)) and the controls (mean bone area, 0.65 +/- 0.67 mm(2)). By eight weeks, the Ad.BMP-2-treated femora had approximately one-fourth of the strength (mean, 0.07 +/- 0.04 Nm) and stiffness (mean, 0.5 +/- 0.4 Nm/rad) of the contralateral femora (0.3 +/- 0.08 Nm and 2.0 +/- 0.5 Nm/rad, respectively). CONCLUSIONS A single, percutaneous, intralesional injection of Ad.BMP-2 induces healing of critical-size femoral bone defects in rats within eight weeks. At this time, the repair tissue is predominantly trabecular bone, has normal bone mineral content, and has gained mechanical strength.

Megakaryocyte-osteoblast interaction revealed in mice deficient in transcription factors GATA-1 and NF-E2

Mice deficient in GATA‐1 or NF‐E2 have a 200–300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6‐fold when cultured with megakaryocytes. A megakaryocyte‐osteoblast interaction plays a role in the increased bone formation in these mice.

Quantitative micro-computed tomography: A non-invasive method to assess equivalent bone mineral density

One of the many applications of micro computed tomography (microCT) is to accurately visualize and quantify cancellous bone microstructure. However, microCT based assessment of bone mineral density has yet to be thoroughly investigated. Specifically, the effects of varying imaging parameters, such as tube voltage (kVp), current (microA), integration time (ms), object to X-ray source distance (mm), projection number, detector array size and imaging media (surrounding the specimen), on the relationship between equivalent tissue density (rhoEQ) and its linear attenuation coefficient (micro) have received little attention. In this study, in house manufactured, hydrogen dipotassium phosphate liquid calibration phantoms (K2HPO4) were employed in addition to a resin embedded hydroxyapatite solid calibration phantoms supplied by Scanco Medical AG Company. Variations in current, integration time and projection number had no effect on the conversion relationship between micro and rhoEQ for the K2HPO4 and Scanco calibration phantoms [p>0.05 for all cases]. However, as expected, variations in scanning tube voltage, object to X-ray source distance, detector array size and imaging media (referring to the solution that surrounds the specimen in the imaging vial) significantly affected the conversion relationship between mu and rhoEQ for K2HPO4 and Scanco calibration phantoms [p<0.05 for all cases]. A multivariate linear regression approach was used to estimate rhoEQ based on attenuation coefficient, tube voltage, object to X-ray source distance, detector array size and imaging media for K2HPO4 liquid calibration phantoms, explaining 90% of the variation in rhoEQ. Furthermore, equivalent density values of bovine cortical bone (converted from attenuation coefficient to equivalent density using the K2HPO4 liquid calibration phantoms) samples highly correlated [R2=0.92] with the ash densities of the samples. In conclusion, Scanco calibration phantoms can be used to assess equivalent bone mineral density; however, they cannot be scanned with a specimen or submerged in a different imaging media. The K2HPO4 liquid calibration phantoms provide a cost effective, easy to prepare and convenient means to perform quantitative microCT analysis using any microCT system, with the ability to choose different imaging media according to study needs. However, as with any liquid calibration phantom, they are susceptible to degradation over time.

Bone Volume Fraction Explains the Variation in Strength and Stiffness of Cancellous Bone Affected by Metastatic Cancer and Osteoporosis

Preventing nontraumatic fractures in millions of patients with osteoporosis or metastatic cancer may significantly reduce the associated morbidity and reduce health-care expenditures incurred by these fractures. Predicting fracture occurrence requires an accurate understanding of the relationship between bone structure and the mechanical properties governing bone fracture that can be readily measured. The aim of this study was to test the hypothesis that a single analytic relationship with either bone tissue mineral density or bone volume fraction (BV/TV) as independent variables could predict the strength and stiffness of normal and pathologic cancellous bone affected by osteoporosis or metastatic cancer. After obtaining institutional review board approval and informed consent, 15 patients underwent excisional biopsy of metastatic prostate, breast, lung, ovarian, or colon cancer from the spine and/or femur to obtain 41 metastatic cancer specimens. In addition, 96 noncancer specimens were excised from 43 age- and site-matched cadavers. All specimens were imaged using micro-computed tomography (micro-CT) and backscatter emission imaging and tested mechanically by uniaxial compression and nanoindentation. The minimum BV/TV, measured using quantitative micro-CT, accounted for 84% of the variation in bone stiffness and strength for all cancellous bone specimens. While relationships relating bone density to strength and stiffness have been derived empirically for normal and osteoporotic bone, these relationships have not been applied to skeletal metastases. This simple analytic relationship will facilitate large-scale screening and prediction of fracture risk for normal and pathologic cancellous bone using clinical CT systems to determine the load capacity of bones altered by metastatic cancer, osteoporosis, or both.

Delayed administration of adenoviral BMP-2 vector improves the formation of bone in osseous defects

The direct, local, administration of adenovirus carrying human BMP-2 cDNA (Ad.BMP-2) heals critical-sized femoral bone defects in rabbit and rat models. However, the outcome is suboptimal and the technology needs to provide a more reliable and uniform outcome. To this end, we investigated whether the timing of Ad.BMP-2 administration influenced the formation of mineralized tissue within the defect. Critical-sized defects were created in the femora of 28 Sprague–Dawley rats. Animals were injected intralesionally with a single, percutaneous injection of Ad.BMP-2 (4 × 108 plaque-forming units) either intraoperatively (day 0) or 24 h (day 1), 5 days or 10 days after surgery. The femora were evaluated 8 weeks after surgery by X-ray, microcomputed tomography, dual-energy X-ray absorptiometry and biomechanical testing. The incidence of radiological union was markedly increased when administration of Ad.BMP-2 was delayed until days 5 and 10, at which point 86% of the defects healed. These time points also provided greater bone mineral content within the defect site and improved the average mechanical strength of the healed bone. Thus, delaying the injection of Ad.BMP-2 until 5 or 10 days after surgery enables a greater percentage of critical-sized, segmental defects to achieve radiological union, producing a repair tissue with enhanced mineralization and greater mechanical strength.

Effects of tissue preservation on murine bone mechanical properties

Murine bone specimens are used extensively in skeletal research to assess the effects of environmental, physiologic and pathologic factors on their mechanical properties. Given the destructive nature of mechanical testing, it is normally performed as a terminal procedure, where specimens must be preserved without affecting their mechanical properties. To this end, we aimed to study the effects of tissue preservation (freezing and formalin fixation) on the elastic and viscoelastic mechanical properties of murine femur and vertebrae. A total of 120 femurs and 180 vertebral bodies (L3-L5) underwent non-destructive cyclic loading to assess their viscoelastic properties followed by mono-cyclic loading to failure to assess their elastic properties. All specimens underwent re-hydration in 0.9% saline for 30min prior to mechanical testing. Analysis indicated that stiffness, modulus of elasticity, yield load, yield strength, ultimate load and ultimate strength of frozen and formalin-fixed femurs and vertebrae were not different from fresh specimens. Cyclic loading of both femurs and vertebrae indicated that loss, storage and dynamic moduli were not affected by freezing. However, formalin fixation altered their viscoelastic properties. Our findings suggest that freezing and formalin fixation over a 2-week period do not alter the elastic mechanical properties of murine femurs and vertebrae, provided that specimens are re-hydrated for at least half an hour prior to testing. However, formalin fixation weakened the viscoelastic properties of murine bone by reducing its ability to dissipate viscous energy. Future studies should address the long-term effects of both formalin fixation and freezing on the mechanical properties of murine bone.

Compressive axial mechanical properties of rat bone as functions of bone volume fraction, apparent density and micro-CT based mineral density

Mechanical testing has been regarded as the gold standard to investigate the effects of pathologies on the structure-function properties of the skeleton. With recent advances in computing power of personal computers, virtual alternatives to mechanical testing are gaining acceptance and use. We have previously introduced such a technique called structural rigidity analysis to assess mechanical strength of skeletal tissue with defects. The application of this technique is predicated upon the use of relationships defining the strength of bone as a function of its density for a given loading mode. We are to apply this technique in rat models to assess their compressive skeletal response subjected to a host of biological and pharmaceutical stimulations. Therefore, the aim of this study is to derive a relationship expressing axial compressive mechanical properties of rat cortical and cancellous bone as a function of equivalent bone mineral density, bone volume fraction or apparent density over a range of normal and pathologic bones. We used bones from normal, ovariectomized and partially nephrectomized animals. All specimens underwent micro-computed tomographic imaging to assess bone morphometric and densitometric indices and uniaxial compression to failure. We obtained univariate relationships describing 71-78% of the mechanical properties of rat cortical and cancellous bone based on equivalent mineral density, bone volume fraction or apparent density over a wide range of density and common skeletal pathologies. The relationships reported in this study can be used in the structural rigidity analysis introduced by the authors to provide a non-invasive method to assess the compressive strength of bones affected by pathology and/or treatment options.

Specimen size and porosity can introduce error into μCT-based tissue mineral density measurements

The accurate measurement of tissue mineral density, rho(m), in specimens of unequal size or quantities of bone mineral using polychromatic microCT systems is important, since studies often compare samples with a range of sizes and bone densities. We assessed the influence of object size on microCT measurements of rho(m) using (1) hydroxyapatite rods (HA), (2) precision-manufactured aluminum foams (AL) simulating trabecular bone structure, and (3) bovine cortical bone cubes (BCt). Two beam-hardening correction (BHC) algorithms, determined using a 200 and 1200 mg/cm(3) HA wedge phantom, were used to calculate rho(m) of the HA and BCt. The 200 mg/cm(3) and an aluminum BHC algorithm were used to calculate the linear attenuation coefficients of the AL foams. Equivalent rho(m) measurements of 500, 1000, and 1500 mg HA/cm(3) rods decreased (r(2)>0.96, p<0.05 for all) as HA rod diameter increased in the 200 mg/cm(3) BHC data. Errors averaged 8.2% across these samples and reached as high as 29.5%. Regression analyses suggested no size effects in the 1200 mg/cm(3) BHC data but differences between successive sizes still reached as high as 13%. The linear attenuation coefficients of the AL foams increased up to approximately 6% with increasing volume fractions (r(2)>0.81, p<0.05 for all) but the strength of the size-related error was also BHC dependent. Equivalent rho(m) values were inversely correlated with BCt cube size (r(2)>0.92, p<0.05). Use of the 1200 mg/cm(3) BHC ameliorated the size-related artifact compared to the 200 mg/cm(3) BHC but errors with this BHC were still significant and ranged between 5% and 12%. These results demonstrate that object size, structure, and BHC algorithm can influence microCT measurements of rho(m). Measurements of rho(m) of specimens of unequal size or quantities of bone mineral must be interpreted with caution unless appropriate steps are taken to minimize these potential artifacts.