Non-invasive prediction of the mouse tibia mechanical properties from microCT images: comparison between different finite element models.

New treatments for bone diseases require testing in animal models before clinical translation, and the mouse tibia is among the most common models. In vivo micro-Computed Tomography (microCT)-based micro-Finite Element (microFE) models can be used for predicting the bone strength non-invasively, after proper validation against experimental data. Different modelling techniques can be used to estimate the bone properties, and the accuracy associated with each is unclear. The aim of this study was to evaluate the ability of different microCT-based microFE models to predict the mechanical properties of the mouse tibia under compressive load. Twenty tibiae were microCT scanned at 10.4 µm voxel size and subsequently compressed at 0.03 mm/s until failure. Stiffness and failure load were measured from the load-displacement curves. Different microFE models were generated from each microCT image, with hexahedral or tetrahedral mesh, and homogeneous or heterogeneous material properties. Prediction accuracy was comparable among models. The best correlations between experimental and predicted mechanical properties, as well as lower errors, were obtained for hexahedral models with homogeneous material properties. Experimental stiffness and predicted stiffness were reasonably well correlated (R2 = 0.53-0.65, average error of 13-17%). A lower correlation was found for failure load (R2 = 0.21-0.48, average error of 9-15%). Experimental and predicted mechanical properties normalized by the total bone mass were strongly correlated (R2 = 0.75-0.80 for stiffness, R2 = 0.55-0.81 for failure load). In conclusion, hexahedral models with homogeneous material properties based on in vivo microCT images were shown to best predict the mechanical properties of the mouse tibia.

Optimization of the failure criterion in micro-Finite Element models of the mouse tibia for the non-invasive prediction of its failure load in preclinical applications.

New treatments against osteoporosis require testing in animal models and the mouse tibia is among the most common studied anatomical sites. In vivo micro-Computed Tomography (microCT) based micro-Finite Element (microFE) models can be used for predicting the bone strength non-invasively, after proper validation against experiments. The aim of this study was to evaluate the ability of different microCT-based bone parameters and microFE models to predict tibial structural mechanical properties in compression. Twenty tibiae were scanned at 10.4 µm voxel size and subsequently tested in uniaxial compression at 0.03 mm/s until failure. Stiffness and failure load were measured from the load-displacement curves. Standard morphometric parameters were measured from the microCT images. The spatial distribution of bone mineral content (BMC) was evaluated by dividing the tibia into 40 regions. MicroFE models were generated by converting each microCT image into a voxel-based mesh with homogeneous isotropic material properties. Failure load was estimated by using different failure criteria, and the optimized parameters were selected by minimising the errors with respect to experimental measurements. Experimental and predicted stiffness were moderately correlated (R2 = 0.65, error = 14% ± 8%). Normalized failure load was best predicted by microFE models (R2 = 0.81, error = 9% ± 6%). Failure load was not correlated to the morphometric parameters and weakly correlated with some geometrical parameters (R2 < 0.37). In conclusion, microFE models can improve the current estimation of the mouse tibia structural properties and in this study an optimal failure criterion has been defined. Since it is a non-invasive method, this approach can be applied longitudinally for evaluating temporal changes in the bone strength.

Experimental evaluation of new chitin-chitosan graft for duraplasty.

Natural materials such as collagen and alginate have promising applications as dural graft substitutes. These materials are able to restore the dural defect and create optimal conditions for the development of connective tissue at the site of injury. A promising material for biomedical applications is chitosan-a linear polysaccharide obtained by the deacetylation of chitin. It has been found to be nontoxic, biodegradable, biofunctional and biocompatible in addition to having antimicrobial characteristics. In this study we designed new chitin-chitosan substitutes for dura mater closure and evaluated their effectiveness and safety. Chitosan films were produced from 3 % of chitosan (molar mass-200, 500 or 700 kDa, deacetylation rate 80-90%) with addition of 20% of chitin. Antimicrobial effictively and cell viability were analysed for the different molar masses of chitosan. The film containing chitosan of molar mass 200 kDa, had the best antimicrobial and biological activity and was successfully used for experimental duraplasty in an in vivo model. In conclusion the chitin-chitosan membrane designed here met the requirements for a dura matter graft exhibiting the ability to support cell growth, inhibit microbial growth and biodegradade at an appropriate rate. Therefore this is a promising material for clinical duroplasty.

Electrospun polyurethane/hydroxyapatite bioactive scaffolds for bone tissue engineering: the role of solvent and hydroxyapatite particles.

Polyurethane (PU) is a promising polymer to support bone-matrix producing cells due to its durability and mechanical resistance. In this study two types of medical grade poly-ether urethanes Z3A1 and Z9A1 and PU-Hydroxyapatite (PU-HA) composites were investigated for their ability to act as a scaffold for tissue engineered bone. PU dissolved in varying concentrations of dimethylformamide (DMF) and tetrahydrofuran (THF) solvents were electrospun to attain scaffolds with randomly orientated non-woven fibres. Bioactive polymeric composite scaffolds were created using 15 wt% Z3A1 in a 70/30 DMF/THF PU solution and incorporating micro- or nano-sized HA particles in a ratio of 3:1 respectively, whilst a 25 wt% Z9A1 PU solution was doped in ratio of 5:1. Chemical properties of the resulting composites were evaluated by FTIR and physical properties by SEM. Tensile mechanical testing was carried out on all electrospun scaffolds. MLO-A5 osteoblastic mouse cells and human embryonic mesenchymal progenitor cells, hES-MPs were seeded on the scaffolds to test their biocompatibility and ability to support mineralised matrix production over a 28 day culture period. Cell viability was assayed by MTT and calcium and collagen deposition by Sirius red and alizarin red respectively. SEM images of both electrospun PU scaffolds and PU-HA composite scaffolds showed differences in fibre morphology with changes in solvent combinations and size of HA particles. Inclusion of THF eliminated the presence of beads in fibres that were present in scaffolds fabricated with 100% DMF solvent, and resulted in fibres with a more uniform morphology and thicker diameters. Mechanical testing demonstrated that the Young׳s Modulus and yield strength was lower at higher THF concentrations. Inclusion of both sizes of HA particles in PU-HA solutions reinforced the scaffolds leading to higher mechanical properties, whilst FTIR characterisation confirmed the presence of HA in all composite scaffolds. Although all scaffolds supported proliferation of both cell types and deposition of calcified matrix, PU-HA composite fibres containing nano-HA enabled the highest cell viability and collagen deposition. These scaffolds have the potential to support bone matrix formation for bone tissue engineering.

Matrix production and collagen structure are enhanced in two types of osteogenic progenitor cells by a simple fluid shear stress stimulus.

Mesenchymal progenitor cells play a vital role in bone regenerative medicine and tissue engineering strategies. To be clinically useful osteoprogenitors should be readily available with the potential to form bone matrix. While mesenchymal stromal cells from bone marrow have shown promise for tissue engineering, they are obtained in small numbers and there is risk of donor site morbidity. Osteogenic progenitor cells derived from dermal tissue may provide a more abundant and easily expandable source of cells. Bone turnover in vivo is regulated by mechanical forces, particularly oscillatory fluid shear stresses (FSS), and in vitro osteogenic progenitors have been shown to be regulated by mechanical stimuli. The aim of this study was to assess what effect osteogenic media and FSS, generated by a simple rocking platform, had on cell behaviour and matrix production in human progenitor dermal fibroblasts (HDFs) and the embryonic stem cell-derived mesenchymal progenitor cell line (hES-MP). Osteogenic media stimulated alkaline phosphatase activity (ALP) and calcium deposition in HDFs. The addition of FSS further enhanced ALP activity and mineralised matrix deposition in both progenitor cells cultured in osteogenic media. Both types of progenitor cell subjected to FSS showed increases in collagen secretion and apparent collagen organisation as imaged by second harmonic generation.

Application of multiple forms of mechanical loading to human osteoblasts reveals increased ATP release in response to fluid flow in 3D cultures and differential regulation of immediate early genes.

ATP is actively released into the extracellular environment from a variety of cell types in response to mechanical stimuli. This is particularly true in bone where mechanically induced ATP release leads to immediate early gene activation to regulate bone remodelling; however there is no consensus as to which mechanical stimuli stimulate osteoblasts the most. To elucidate which specific type(s) of mechanical stimuli induce ATP release and gene activation in human osteoblasts, we performed an array of experiments using different mechanical stimuli applied to both monolayer and 3D cultures of the same osteoblast cell type, SaOS-2. ATP release from osteoblasts cultured in monolayer significantly increased in response to turbulent fluid flow, laminar fluid flow and substrate strain. No significant change in ATP release could be detected in 3D osteoblast cultures in response to cyclic or static compressive loading of osteoblast-seeded scaffolds, whilst turbulent fluid flow increased ATP release from 3D cultures of osteoblasts to a greater degree than observed in monolayer cultures. Cox-2 expression quantified using real time PCR was significantly lower in cells subjected to turbulent fluid flow whereas c-fos expression was significantly higher in cells subjected to strain. Load-induced signalling via c-fos was further investigated using a SaOS-2 c-fos luciferase reporter cell line and increased in response to substrate strain and turbulent fluid flow in both monolayer and 3D, with no significant change in response to laminar fluid flow or 3D compressive loading. The results of this study demonstrate for the first time strain-induced ATP release from osteoblasts and that turbulent fluid flow in 3D up regulates the signals required for bone remodelling.

Short bouts of mechanical loading are as effective as dexamethasone at inducing matrix production by human bone marrow mesenchymal stem cell.

Dexamethasone (Dex) is used widely to induce differentiation in human mesenchymal stem cells (hMSCs); however, using a pharmaceutical agent to stimulate hMSC differentiation is not the best choice for engineered tissue transplantation due to potential side-effects. The goal of the present study was to investigate the effects of dynamic compressive loading on differentiation and mineralized matrix production of hMSCs in 3D polyurethane scaffolds, using a loading regimen previously shown to stimulate mineralised matrix production of mature bone cells (MLO-A5). hMSCs were seeded in polyurethane scaffolds and cultured in standard culture media with or without Dex. Cell-seeded scaffolds were compressed at 5% global strain for 2 h on day 9 and then every 5 days in a media-filled sterile chamber. Samples were tested for mRNA expression of alkaline phosphatase (ALP), osteopontin (OPN), collagen type 1 (col 1) and runt-related transcription factor-2 (RUNX-212 h) after the first loading, cell viability by MTS assay and alkaline phosphatase activity at day 12 of culture and cell viability, collagen content by Sirius red and calcium content by alizarin red at day 24 of culture. Neither Dex nor loading had significant effects on cell viability. Collagen content was significantly higher (p<0.01) in the loaded group compared with the non-loaded group in all conditions. There was no difference in ALP activity or the amount of collagen and calcium produced between the non-loaded group supplemented with Dex and the loaded group without Dex. We conclude that dynamic loading has the ability to stimulate osteogenic differentiation of hMSC in the absence of glucocorticoids.

Mechanisms of fluid-flow-induced matrix production in bone tissue engineering.

Matrix production by tissue-engineered bone is enhanced when the growing tissue is subjected to mechanical forces and/or fluid flow in bioreactor culture. Cells deposit collagen and mineral, depending upon the mechanical loading that they receive. However, the molecular mechanisms of flow-induced signal transduction in bone are poorly understood. The hyaluronan (HA) glycocalyx has been proposed as a potential mediator of mechanical forces in bone. Using a parallel-plate flow chamber the effects of removal of HA on flow-induced collagen production and NF-kappaB activation in MLO-A5 osteoid osteocytes were investigated. Short periods of fluid flow significantly increased collagen production and induced translocation of the NF-kappaB subunit p65 to the cell's nuclei in 65 per cent of the cell population. Enzymatic removal of the HA coat and antibody blocking of CD44 (a transmembrane protein that binds to HA) eliminated the fluid-flow-induced increase in collagen production but had no effect on the translocation of p65. HA and CD44 appear to play roles in transducing the flow signals that modulate collagen production over long-term culture but not in the short-term flow-induced activation of NF-kappaB, implying that multiple signalling events are initiated from the commencement of flow. Understanding the mechanotransduction events that enable fluid flow to stimulate bone matrix production will allow the optimization of bioreactor design and flow profiles for bone tissue engineering.

Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts.

Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca(2+)i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3-E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to -2 Newtons/m(2) at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca(2+)i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca(2+)i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway.

Investigation of the morphology of the lacunocanalicular system of cortical bone using atomic force microscopy.

Mechanical loading has been implicated as a powerful driving mechanism for interstitial fluid flow through bone. However, little information is available with regard to the morphology of bone fluid spaces, e.g., the canalicular wall, which would be expected to dictate the type of flow regime developing in the lacunocanalicular system under mechanical loads. The purpose of this study was to examine the fine structure of the lacunocanalicular system in cortical bone using atomic force microscopy (AFM), resin casting methods, and selective etching of the specimen surface. A resin-cast replica of the canalicular wall was produced and surface morphology and dimensions were observed using AFM in tapping mode. Material contrast was obtained using surface potential measurements. A striped pattern perpendicular to the canaliculus long axis with a periodicity of 125 nm dominated the structure of the canalicular wall; it is likely that this was caused by the imprint of collagen fibrils arranged in parallel, lining the canaliculus wall. The largest dimension measured for canalicular diameter was on the order of 500 nm. The regular dips and ridges caused by the collagen that lines the wall are a source of roughness which may influence shear stresses imparted by the fluid on the cell surface as well as mixing of solutes within the lacunocanalicular system. In addition, the lacunocanalicular wall lining is likely to affect physicochemical interactions between the fluid and bone matrix. This has important implications for modeling and understanding the microfluid mechanics and rheology of the fluid-filled lacunocanalicular network.

Karyotyping for isolated neural tube defects. A report of two cases.

BACKGROUND: Neural tube defects occur in approximately 1 in every 1,000 live births. In the United States, chromosomal abnormalities have been noted in 2-10% of fetuses with neural tube defects; however, there is no consensus on whether to offer karyotype analysis to patients with isolated neural tube defects found on ultrasound. CASE: We reviewed the prenatal diagnosis database for the University of Washington between 1985 and 1997. We report on two fetuses who, on ultrasound, were found to have "isolated" neural tube defects. Karyotype analysis revealed trisomy 18 in both fetuses. The pregnancies were subsequently terminated, and autopsy revealed subtle syndromic findings that were not identified on ultrasound. CONCLUSION: Fetuses with isolated neural tube defects also appear to have a high risk of chromosomal abnormalities, so patients should be offered fetal karyotyping to define recurrence risks for future pregnancies.

Observations of microdamage around osteocyte lacunae in bone.

Tensile microdamage was examined using laser scanning confocal microscopy in beam specimens of bovine, equine and human long bones loaded in vitro and whole specimens of rat ulnae loaded in vivo. Microcracks were observed to initiate frequently at osteocyte lacunae. The implication is that osteocyte lacunae act as stress concentrating features in bone. This association provides a potential mechanism for the detection of strain and/ or damage by osteocytes in bone.

Postexercise and positional variation in mechanical properties of the radius in young horses.

The metacarpal of the horse is severely loaded during vigorous exercise. Metacarpal specimens have a greater impact strength in young horses that have been exercised than in those that have only been walked. We did not find a corresponding difference in the radius of the same horses. We show that cranial (anterior) cortical bone from the radius, which is loaded in tension during locomotion, has a greater Young's modulus, and tensile and bending strength, than bone from the caudal (posterior) cortex, which is loaded in compression. Caudal bone is, however, stronger in compression. The differences can be explained by differences in the histological structure developed by the 2 cortices and are presumably adaptive. This work confirms the work of others. Furthermore, we demonstrate that the impact energy absorption of cranial bone is nearly twice as great as that of caudal bone. The caudal cortex has apparently paid a heavy price in its reduction in resistance to accidental impact loading for being stronger than the cranial cortex in compressive loading.

The effects of damage and microcracking on the impact strength of bone.

Microcracking has been shown to occur when bone is 'damaged' as shown by a loss of stiffness. The effect on bone's toughness of the types of damage produced at low losses of stiffness are not known. We loaded bovine bone specimens in bending and tension to stiffness losses of up to 27%, and examined the microcracking produced. The tensile specimens had diffuse arrays of microcracks of 2-20 microm in length, characteristic of tensile loading, on all surfaces. The bending specimens showed tensile microcracking on the tensile surface and characteristic long, straight, cross-hatched compression cracks on the compressive surface. Specimens were then broken in impact. Those that had been damaged in bending were divided into two groups, in one group the part of the specimen which had undergone compression damage was placed in tension, and in the other group the tensile damage was placed in tension. Tensile damage loaded in tension did not reduce the bone's energy-absorbing ability in impact until a modulus reduction of over 20%. However compression damage loaded in tension did severely reduce the bone's energy absorption capabilities (by an average of about 40%).

Maternal second trimester serum tumor necrosis factor-alpha-soluble receptor p55 (sTNFp55) and subsequent risk of preeclampsia.

Preeclampsia is characterized by diffuse vascular endothelial dysfunction. Tumor necrosis factor-alpha (TNF-alpha), which plays a key role in the cytokine network responsible for immunoregulation, is also known to contribute to endothelial dysfunction and other metabolic disturbances noted in preeclampsia. Results from cross-sectional studies and one longitudinal study indicate that TNF-alpha (or its soluble receptor, sTNFp55) is increased in the peripheral circulation and amniotic fluid of women with preeclampsia as compared with normotensive women. Between December 1993 and August 1994, prediagnostic sTNFp55 concentrations (a marker of excessive TNF-alpha release) were measured in 35 women with preeclampsia and 222 normotensive women to determine whether elevations precede the clinical manifestation of the disorder. Logistic regression procedures were used to calculate maximum likelihood estimates of odds ratios and 95% confidence intervals. Mean second trimester (15-22 weeks' gestation) serum sTNFp55 concentrations, measured by enzyme-linked immunosorbent assay, were 14.4% higher in preeclamptic women than in normotensive controls (716.6 pg/ml (standard deviation 193.6) vs. 626.4 pg/ml (standard deviation 158.0); p = 0.003). The relative risk of preeclampsia increased across successively higher quintiles of sTNFp55 (odds ratios were 1.0, 1.3, 2.1, and 3.7, with the lowest quintile used as the referent; p for trend = 0.007). After adjustment for maternal age, adiposity, and parity, the relative risk between extreme quintiles was 3.3 (95% confidence interval 0.8-13.4). These findings indicate that the level of TNF-alpha in maternal circulation is increased prior to the clinical manifestation of the disorder, and they are consistent with the hypothesized role of cytokines in mediating endothelial dysfunction and the pathogenesis of preeclampsia. Further work is needed to identify modifiable risk factors for the excessive synthesis and release of TNF-alpha in pregnancy, and to assess whether lowering of TNF-alpha concentrations in pregnancy alters the incidence and severity of preeclampsia.

The development of microcracking and failure in bone depends on the loading mode to which it is adapted.

During locomotion, the anterior cortex of the equine radius is loaded predominantly in tension, the posterior predominantly in compression. The anterior cortex is relatively strong in tension, the posterior in compression. We investigated the pattern of failure of specimens from the two cortices using laser scanning confocal microscopy. All specimens were loaded in four-point bending to increasingly higher loads. We quantified the amount of diffuse microcracking on the tensile side of these specimens by observing the amount of light emitted under laser illumination. The amount of light emitted agreed well with subjective estimates of the amount of microcracking. Tensile microcracks first appeared at a strain of approximately 0.004, and all specimens showed considerable growth in microcrack density once the tensile strain had passed approximately 0.008. In specimens from the posterior cortex, there was little compressive microcracking, and such cracks as were present were small and diffuse. These specimens failed on the tensile side first. In specimens from the anterior cortex, compression cracks were more numerous, longer and less diffuse, and specimens failed initially in compression. The patterns of failure in the bone tissues of the two cortices are what would be expected assuming they were adapted to the mode of loading to which they are usually subjected.

Effects of ionizing radiation on the mechanical properties of human bone.

Allogeneic bone grafts are frequently sterilized by means of ionizing radiation. We investigated the effects of ionizing radiation on both quasistatic and impact mechanical properties of human bone. Specimens from four paired femora of four donors received doses of 29.5 kGy ("standard," frequently used by tissue banks), 94.7 kGy ("high"), or 17 kGy ("low") of ionizing radiation. Young's modulus was unchanged by any level of radiation. Radiation significantly reduced bending strength, work to fracture, and impact energy absorption; in each case, the severity of the effect increased from low to standard to high doses of radiation. Work to fracture was particularly severely degraded; specimens irradiated with the high dose absorbed only 5% of the energy of the controls. Radiation, even at relatively low doses, makes the bone more brittle and thereby reduces its energy-absorbing capacity. We suggest that because the level of radiation required to produce an acceptable level of viral inactivation (90 kGy) produces an unacceptable reduction in the mechanical integrity of the bone, low levels of radiation, sufficient to produce bacterial safety, should be used in conjunction with biological tests to ensure viral safety.

Exercise of young thoroughbred horses increases impact strength of the third metacarpal bone.

Exercise can have a profound effect on bone mass, but little is known of its effect on bone's material properties. In this experiment, our hypothesis was that a large difference in the training regimen of young thoroughbreds would produce a measurable difference in the mechanical properties of their bone material. When they were about 19 months old, eight thoroughbred racehorses were given one of two exercise regimens that lasted for 19 weeks: four horses (controls) were walked for 40 minutes a day but had no other exercise, and the remaining four (exercised) were additionally trotted for 20 minutes a day and given progressively intensive exercise on a treadmill. Mechanical testing to failure was performed on longitudinal beam specimens of the mid-diaphysis of the metacarpal. There was no difference in Young's modulus or bending strength between the two groups, although these properties varied somewhat depending on the position within the cortex from which the specimens had come. The specimens from the exercised horses had a slightly higher toughness, as measured by work (area under the load-deformation curve). They had a considerably higher impact strength. The impact strength of specimens from the outer cortex was also higher than that of those from the inner cortex in both groups. Impact strength correlated positively with the amount of microcracking produced during testing. Microcracking is related to structural and microstructural features in the bone. Increased loading caused the bone to respond in a way that enhanced its ability to microcrack and hence its toughness.

Effect of formaldehyde fixation on some mechanical properties of bovine bone.

The risk of infection of investigators working on the biomechanics of human bone from a variety of modern pathogens including the human immunodeficiency virus or the hepatitis B virus has increased recently. New safety procedures are needed to reduce that risk. The procedure we follow in our laboratory employs brief (< 3 h) fixation in formaldehyde, and we report here the effects it has on some mechanical properties of bovine bone. Results in quasistatic loading tests were almost unaffected by our fixation protocol, but a significant decrease in impact strength was found. These results indicate that there may be some interaction between fixation and strain rate dependent effects and, therefore, some caution is needed when using common biomechanical measurement methods on fixed bone material.