In situ visualization of aortic dissection propagation in notched rabbit aorta using synchrotron X-ray tomography.

Aortic dissection is a complex, intramural, and dynamic condition involving multiple mechanisms, hence, difficult to observe. In the present study, a controlled in vitro aortic dissection was performed using tension-inflation tests on notched rabbit aortic segments. The mechanical test was combined with conventional (cCT) and synchrotron (sCT) computed tomography for in situ imaging of the macro- and micro-structural morphological changes of the aortic wall during dissection. We demonstrate that the morphology of the notch and the aorta can be quantified in situ at different steps of the aortic dissection, and that the notch geometry correlates with the critical pressure. The phenomena prior to propagation of the notch are also described, for instance the presence of a bulge at the tip of the notch is identified, deforming the remaining wall. Finally, our method allows us to visualize for the first time the propagation of an aortic dissection in real-time with a resolution that has never previously been reached. STATEMENT OF SIGNIFICANCE: With the present study, we investigated the factors leading to the propagation of aortic dissection by reproducing this mechanical process in notched rabbit aortas. Synchrotron CT provided the first visualisation in real-time of an aortic dissection propagation with a resolution that has never previously been reached. The morphology of the intimal tear and aorta was quantified at different steps of the aortic dissection, demonstrating that the early notch geometry correlates with the critical pressure. This quantification is crucial for the development of better criteria identifying patients at risk. Phenomena prior to tear propagation were also described, such as the presence of a bulge at the tip of the notch, deforming the remaining wall.

Deletion of osteopontin or bone sialoprotein induces opposite bone responses to mechanical stimulation in mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP) are co-expressed in bone and display overlapping and complementary physiological properties. Both genes show a rapid expression response to mechanical stimulation. We used mice with single and double deletions (DKO) of BSP and OPN to assess the specificity of their roles in skeletal adaptation to loading. Two-month-old Wild-Type (WT), BSP knockout (BSP-/-), OPN-/- and DKO male mice were submitted to two mechanical stimulation regimen (n = 10 mice/group) respectively impacting trabecular bone (Hypergravity, HG) and cortical bone (Whole Body Vibration, WBV). HG increased trabecular bone volume (BV/TV) in WT femur through reduced resorption, and in BSP-/- mice femur and vertebra through increased bone formation. In contrast, HG increased the turnover of OPN-/- bone, resulting in reduced femur and vertebra BV/TV. HG did not affect DKO bones. Similarly, WBV increased cortical thickness in BSP-/- mice and decreased it in OPN-/-, without affecting structurally WT and DKO bone. Vibrated BSP-/- mice displayed increased endocortical bone formation with a drop in Sclerostin expression, and reduced periosteal osteoclasts with lower Rankl and Cathepsin K expression. In contrast, vibrated OPN-/- endocortical bone displayed decreased formation and increased osteoclast coverage. Therefore, under two regimen (HG and WBV) targeting distinct bone compartments, absence of OPN resulted in bone loss while lack of BSP induced bone gain, reflecting divergent structural adaptations. Strikingly, absence of both proteins led to a relative insensitivity to either mechanical challenge. Interplay between OPN and BSP thus appears as a key element of skeletal response to mechanical stimulation.

Deletion of OPN in BSP knockout mice does not correct bone hypomineralization but results in high bone turnover.

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.

Structure of the cortical cytoskeleton in fibers of postural muscles and cardiomyocytes of mice after 30-day 2-g centrifugation.

Altered external mechanical loading during spaceflights causes negative effects on muscular and cardiovascular systems. The aim of the study was estimation of the cortical cytoskeleton statement of the skeletal muscle cells and cardiomyocytes. The state of the cortical cytoskeleton in C57BL6J mice soleus, tibialis anterior muscle fibers, and left ventricle cardiomyocytes was investigated after 30-day 2-g centrifugation ("2-g" group) and within 12 h after its completion ("2-g + 12-h" group). We used atomic force microscopy for estimating cell's transverse stiffness, Western blotting for measuring protein content, and RT-PCR for estimating their expression level. The transverse stiffness significantly decreased in cardiomyocytes (by 16%) and increased in skeletal muscles fibers (by 35% for soleus and by 29% for tibialis anterior muscle fibers) in animals of the 2-g group (compared with the control group). For cardiomyocytes, we found that, in the 2-g + 12-h group, α-actinin-1 content decreased in the membranous fraction (by 27%) and increased in cytoplasmic fraction (by 28%) of proteins (compared with the levels in the 2-g group). But for skeletal muscle fibers, similar changes were noted for α-actinin-4, but not for α-actinin-1. In conclusion, we showed that the different isoforms of α-actinins dissociate from cortical cytoskeleton under increased/decreased of mechanical load.

Vaginal self-sampling is a cost-effective way to increase participation in a cervical cancer screening programme: a randomised trial.

BACKGROUND: Cervical cancer screening coverage remains insufficient in most countries. Our objective was to assess whether in-home vaginal self-sampling with a dry swab for high-risk human papillomavirus (HR-HPV) testing is effective and cost-effective in increasing participation in cervical cancer screening. METHODS: In March 2012, 6000 unscreened women aged 30-65 years, living in a French region covered by a screening programme, who had not responded to an initial invitation to have a Pap smear were equally randomised to three groups: 'no intervention'; 'recall', women received a letter to have a Pap smear; and 'self-sampling', women received a self-sampling kit to return to a centralised virology laboratory for PCR-based HPV testing. RESULTS: Participation was higher in the 'self-sampling' than in the 'no intervention' group (22.5% vs 9.9%, P<0.0001; OR 2.64) and 'recall' group (11.7%, P<0.0001; OR 2.20). In the 'self-sampling' group, 320 used the self-sampling kit; for 44 of these women with positive HR-HPV test results, 40 had the recommended triage Pap smear. The ICER per extra screened woman was 77.8[euro ] and 63.2[euro ] for the 'recall' and 'self-sampling' groups, respectively, relative to the 'no intervention' group. CONCLUSIONS: Offering an in-home, return-mail kit for vaginal self-sampling with a dry swab is more effective and cost-effective than a recall letter in increasing participation in cervical cancer screening.

A systematic review of methods for tissue analysis in animal studies on orthodontic mini-implants.

Anchorage devices are increasingly used in orthodontics, and their clinical performance is directly dependent on the tissue response to these devices. This study aims to identify assessment parameters for evaluating tissue reactions around orthodontically loaded implants and to propose parameters to be included in a standardized method. Several electronic databases (PubMed, ScienceDirect, the Cochrane database) were explored for papers from January 1999 to December 2009. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement was used as a guideline for the methodology of systematic reviews. Twenty-five publications were selected from 123 potentially relevant abstracts. The selected studies mainly aimed to answer a clinical question and particularly the ability of immediate loading in orthodontics. Very few studies aimed to understand the healing mechanism around the devices leading to a lack of information on this topic. The most frequent combination of assessment methods was clinical evaluation, histology/histomorphometry and intravital bone labeling. Although the dog model is mainly used, pigs represent an interesting animal model, especially when studying devices in growing bone. Despite the extensive use of miniscrews in growing individuals, only few studies have included young subjects in their protocol. Moreover, in such studies, an oral hygiene program is absolutely necessary to avoid complications. Finite element analysis could improve the knowledge of the relationship between design and bone reaction; unfortunately, this elaborated method is complex and impossible to perform routinely. For standardization, the authors recommend to include specific criteria in study protocols when assessing tissue response to orthodontically loaded devices.

Reduction by strontium of the bone marrow adiposity in mice and repression of the adipogenic commitment of multipotent C3H10T1/2 cells.

Multipotent mesenchymal cells (MMCs) differentiate into osteoblasts or adipocytes through RUNX2 and PPARγ2, respectively. Strontium ranelate has been shown to promote osteoblastogenesis and prevent adipogenesis in long-term experiments using MMCs. The present study involved in-vitro and in-vivo investigations of whether Sr might first be an inhibitor of adipogenesis, thus explaining late osteoblastogenesis. It was established in vivo that Sr reduces adipogenesis in mice treated only for 3 weeks with a 6 mmol/kg/day dose of Sr while the trabecular bone volume is increased. In order to decipher molecular mechanisms during inhibition of adipogenesis, we used murine MMCs C3H10T1/2 cultured under adipogenic conditions (AD) and treated Sr of a concentration up to 3 mM. It was shown that early on (day 1), Sr dose-dependently reduced PPARγ2 and CEBPα mRNA without affecting the RUNX2 gene expression whereas it repressed ALP mRNA. Later (day 5), PPARγ2 and CEBPα mRNA remained inhibited by Sr, preventing adipocyte lipid accumulation, while Runx2 and ALP mRNA were increased. Moreover, under the mentioned conditions, Sr was able to quickly induce the Cyclin D1 gene expression, proliferation and fibronectin fibrillogenesis, both involved in the inhibition of adipogenesis. The inhibition of the ERK pathway by U0126 blunted the Sr-induced PPARγ2 repression while restoring the lipid accumulation. These results demonstrated that Sr was capable of rapidly reducing adipogenesis by a selective PPARγ2 repression that can be explained by its ability to promote MMC proliferation.

New insight into the bony labyrinth: a microcomputed tomography study.

OBJECTIVES: To visualize and quantify the morphology and mineralization of the developing fetal human bony labyrinth, using 3D-microcomputed tomography (3D-microCT) imaging. METHODS: Eleven right temporal bones from late second and third trimester fetuses were used in this prospective pilot study. After fixation in 10% formalin solution, all samples underwent a microcomputed tomography (microCT) scan, permitting the 3D imaging of the bony labyrinth as well as the quantitative assessment of mineral density, angular distances and dimensions of inner ear components the progression of ossification was precised with histological observations. RESULTS: Our findings show different rates of growth among the semicircular canals, the vestibular aqueduct, the oval window, the round window and the cochlea. The final sizes of the cochlea and round window are achieved at 23 weeks of gestation, with heights of 5mm and 2mm, respectively. The oval window reaches adult size at 35 weeks, whereas the vestibular aqueduct will attain adult size after birth. An increasing degree of torsion of each semicircular canal is observed during fetal development. The superior semicircular canal achieves adult size at 24 weeks, before the posterior and the lateral canals (25 weeks). The time-course of ossification and mineralization observed in structures and confirmed by histology. CONCLUSIONS: During this developmental period poorly studied until now, our findings suggest that each part of the bony labyrinth follows distinct growth and ossification kinetics trajectories, some of these reaching their adult size only after birth.

Assessment of trabecular bone microarchitecture by two different x-ray microcomputed tomographs: a comparative study of the rat distal tibia using Skyscan and Scanco devices.

Important aspects of modern skeletal research depend on the phenotypical characterization of trabecular bone microarchitecture as assessed by microcomputed tomography (micro-CT). Until now, however, there have been no studies which compare the two most commonly utilized micro-CT devices, namely, Skyscan and Scanco. The purpose of the current study was to examine the reproducibility and accuracy of these two micro-CT devices in comparison to traditional histomorphometry in ovariectomized rats treated with either propranolol, salbutamol, or vehicle. 6 month old female Wistar rats were ovariectomized (n = 48) or sham operated (n = 12). OVX rats were divided into four groups and then subcutaneously injected with propranolol 0.1 mg/kg/day, propranolol 20 mg/kg/day, salbutamol 3 mg/kg/day, or vehicle for 10 weeks. At sacrifice, the left tibial trabecular bone microarchitecture was analyzed using both the micro-CT Skyscan 1072 (ex vivo) and Scanco vivaCT40 (in vivo). Histomorphometric analysis was performed on the right proximal tibia. Comparisons between the different methods were performed using regression analysis, Bland-Altman, Passing-Bablock, and Mountain plots. Correlations were highly significant for all parameters measured between the two micro-CT instruments and were less significant between histomorphometry and micro-CT measurements taken from either the Skyscan or Scanco apparatus. Micro-CT overestimated bone volume compared to histomorphometry. In the ovariectomized rat model, the two micro-CT instruments revealed the same difference between groups whereas histomorphometry revealed only the difference which displayed the largest disparity between groups. In regards to the comparison between the two micro-CT devices, Mountain plot methods indicated that BV/TV, BS/BV, and TbSp were equivalent, whereas a systematic bias was observed for TbN and TbTh. The authors were also able to describe the routine method used to determine the threshold between the two micro-CT devices, which may help explain these differences. While some minor differences in the absolute values of the morphometry parameters exist between the micro-CT measurements from the Skyscan and Scanco instruments, both of these devices display a high degree of accuracy and reproducibility.

Doping dose of salbutamol and exercise training: impact on the skeleton of ovariectomized rats.

Previous studies in healthy rats have demonstrated a deleterious bone impact of beta-agonist treatment. The purpose of this study was to examine the trabecular and cortical effects of beta(2)-agonists at doping dose on treadmill exercising rats with estrogen deficiency. Adult female rats were ovariectomized (OVX; n = 44) or sham operated (n = 12). Then, OVX rats received a subcutaneous injection of salbutamol (SAB) or vehicle with (EXE) or without treadmill exercise for 10 wk. Bone mineral density (BMD) was analyzed by densitometry. Microcomputed tomography and histomorphometric analysis were performed to study trabecular bone structure and bone cell activities. After 10 wk, SAB rats presented a much more marked decrease of BMD and trabecular parameters. Exercise did not change the high level of bone resorption in OVX EXE SAB compared with OVX SAB group (both on COOH-terminal collagen cross-links and osteoclast number). These results confirm the deleterious effect of beta(2)-agonists on bone quantity (femoral BMD gain: OVX EXE, +6.8%, vs. OVX EXE SAB, -1.8%; P < 0.01) and quality (-8.0% of femoral trabecular thickness in OVX EXE SAB vs. OVX EXE), indicating that SAB suppresses the effect of EXE in OVX rats. Furthermore, we notice that the slight beneficial effect of exercise was mainly localized in the tibia. These findings indicate the presence of a bone alteration threshold below which there is no more alteration in structural bone quantity and quality. The negative effects of SAB on bone observed in this study in trained rats may indicate potential complications in doping female athletes with exercise-induced amenorrhea.

Doping dose of salbutamol and exercise: deleterious effect on cancellous and cortical bones in adult rats.

Animal studies suggest that bone remodeling is under beta-adrenergic control via the sympathetic nervous system. To our knowledge, the impact of beta-agonist substances, at doping doses, has not been studied in adult rats. The purpose of this study was to examine the effects of salbutamol injections with or without treadmill exercise on trabecular and cortical bone in adult rats. Adult (36 wk of age) female Wistar rats (n = 56) were treated with salbutamol (3 mg.kg(-1).day(-1) sc, 5 days/wk) or vehicle (sham) with or without subsequent treadmill exercise (13 m/min, 60 min/day, 5 days/wk) for 10 wk. Tibial and femoral bone mineral density was analyzed by dual-energy X-ray absorptiometry. Metaphysic trabecular bone structure was analyzed by micro-CT at the time of the animals' death. Bone cell activities were assessed histomorphometrically. After 10 wk, the increase in bone mineral density was less in salbutamol-treated than in sham rats (+3.3% vs. +12.4%, P < 0.05), and trabecular parameters were altered and bone resorption was increased in salbutamol-treated rats compared with controls. The negative effect on bone architecture in salbutamol-treated rats persisted, even with treadmill exercise. These results confirm the deleterious effect of beta(2)-agonists on bone mass during chronic treatment and describe its effects on bone mechanical properties in adult rats. Bone loss occurred independently of a salbutamol-induced anabolic effect on muscle mass and was equally severe in sedentary and exercising rats, despite a beneficial effect of exercise on the extrinsic and intrinsic energy to ultimate strain. These bone effects may have important consequences in athletes who use salbutamol as a doping substance.

Dose effects of propranolol on cancellous and cortical bone in ovariectomized adult rats.

Animal studies suggest that bone remodeling is under beta-adrenergic control via the sympathetic nervous system. The purpose of this study was to examine the preventive effect of different doses of nonspecific beta-blockers (propranolol) on trabecular and cortical bone envelopes in ovariectomized rats. Six-month-old female Wistar rats were ovariectomized (OVX, n = 60) or sham-operated (n = 15). Then, OVX rats were subcutaneously injected with 0.1 (n = 15), 5 (n = 15), or 20 (n = 15) mg/kg propranolol or vehicle (n = 15) for 10 weeks. Tibial and femoral bone mineral density (BMD) were analyzed longitudinally by dual-energy X-ray absorptiometry. At death, the left tibial metaphysis and L(4) vertebrae were removed, and microcomputed tomography (Skyscan 1072; Skyscan, Aartselaar, Belgium) was performed for trabecular bone structure investigation. Histomorphometry analysis was performed on the right proximal tibia to assess bone cell activities. After 10 weeks, OVX rats had decreased BMD and trabecular parameters and increased bone turnover, as well as cortical porosity compared with the sham group (p < 0.001). Bone architecture alteration was preserved by 0.1 mg/kg propranolol due to higher trabecular number and thickness (+50.35 and +6.81%, respectively, than OVX; p < 0.001) and lower cortical pore number (-52.38% than OVX; p < 0.001). Animals treated by 0.1 mg/kg propranolol had a lower osteoclast surface and a higher osteoblast activity compared with OVX. Animals treated by 20 mg of propranolol did not significantly differ from OVX rats. Animals treated by 5 mg of propranolol have been partially preserved from the ovariectomy. These results showed a dose effect of beta-blockers. The lower the dose of propranolol breeding, the better the preventive effect against ovariectomy.

Dramatic decrease of innervation density in bone after ovariectomy.

Recent studies have demonstrated that bone is highly innervated and contains neuromediators that have functional receptors on bone cells. However, no data exist concerning the quantitative changes of innervation during bone loss associated with estrogen withdrawal. To study the involvement of nerve fibers in the regulation of bone remodeling, we have evaluated the modifications of innervation in a classical in vivo model of osteopenia in rats, ovariectomy (OVX). Skeletal innervation was studied by immunocytochemistry using antibodies directed against specific neuronal markers, neurofilament 200 and synaptophysin, and the neuromediator glutamate. Sciatic neurectomy, another model of bone loss due to limb denervation and paralysis, was used to validate our quantitative image analysis technique of immunostaining for nerve markers. Female Wistar rats at 12 wk of age were sham-operated (SHAM) or ovariectomized (OVX). Bone mineral density measurement and bone histomorphometry analysis of tibiae 14 d after surgery demonstrated a significant bone loss in OVX compared with SHAM. We observed an important reduction of nerve profile density in tibiae of OVX animals compared with SHAM animals, whereas innervation density in skin and muscles was similar for OVX and control rats. Quantitative image analysis of immunostainings demonstrated a significant decrease of the percentage of immunolabeling per total bone volume of neurofilament 200, synaptophysin, and glutamate in both the primary and secondary spongiosa of OVX rats compared with SHAM. These data indicate for the first time that OVX-induced bone loss in rat tibiae is associated with a reduction in nerve profile density, suggesting a functional link between the nervous system and the bone loss after ovariectomy.

Cell cycling determines integrin-mediated adhesion in osteoblastic ROS 17/2.8 cells exposed to space-related conditions.

Six days of microgravity (Bion10 mission) induced dramatic shape changes in ROS 17/2.8 osteoblasts (7). During the Foton 11 and 12 space flights, we studied the kinetics (0-4 days) of ROS 17/2.8 morphology and adhesion, the relationships between adhesion and cell cycle progression after 4 days in space, and osteoblastic growth and activity after 6 days in space. Quantitative analysis of high-resolution adhesion [focal adhesion area imaged by total interference reflection fluorescent microscopy (TIRFM)] and integrin-dependent adhesion (imaged on confocal microscope by vinculin and phosphotyrosine staining) as well as cell cycle phase classification [Ki-67 staining, S-G2, mitotic cells and G1 (postmitotic cells)] were performed using programs validated in parabolic flight and clinostat. We observed disorganization of the cytoskeleton associated with disassembling of vinculin spots and phosphorylated proteins within focal contacts with no major change in TIRFM adhesion after 2 and 4 days of microgravity. Postmitotic cells, alone, accounted for the differences observed in the whole population. They are characterized by immature peripheral contacts with complete loss of central spots and decreased spreading. Osteocalcin, P1CP and alkaline phosphatase, and proliferation were similar in flight cells and 1 g centrifuge and ground controls after 6 days. In conclusion, microgravity substantially affected osteoblastic integrin-mediated cell adhesion. ROS17/2.8 cells responded differently, whether or not they were cycling by reorganizing adhesion plaque topography or morphology. In ROS 17/2.8, this reorganization did not impair osteoblastic phenotype.

Various evaluation techniques of newly formed bone in porous hydroxyapatite loaded with human bone marrow cells implanted in an extra-osseous site.

The purpose of this work was to develop qualitative methods for in situ analysis of bone formation in an osteoconductive hydroxyapatite matrix (ENDOBON), loaded with human bone marrow cells (HBMSC) implanted subcutaneously in athymic mice. Samples were taken before implantation (T0), 1, 2, 4 and 6 weeks after implantation. Bone-biomaterial interaction were investigated on undecalcified sections by histological, cytochemical, immunological and molecular biology methodologies. Histological observations were performed in order to observe inflammatory cells, vessels, newly formed bone, woven and lamellar bone. Enzymohistochemistry was carried out to detect positive tartrate resistant acid phosphatase activity (TRAP+). Immunohistochemistry using antibodies against type I collagen and osteocalcin permitted us to characterize the content of the matrix elaborated within the implant. Moreover, in situ hybridization was carried out to discriminate, the implanted human cells from the murine cells, and to evaluate the function of these human cells in osteogenesis. Results demonstrated an early formation of lamellar bone only in the pores of the studied HAP loaded with HBMSC. This bone contained a matrix showing positive reaction for type I collagen and osteocalcin. In situ hybridization identified some of these cells as human cells. At 6 weeks, examination of histological results showed persistance of lamellar bone in the implants. We only found TRAP+ activity in the materials loaded with human bone marrow cells. Molecular hybridization no longer revealed positive cells for the human DNA probe. All these results indicate that the various evaluation techniques performed on undecalcified sections, permit us to evaluate the response of human bone marrow cells in HAP implanted into mice.

Effects of centrifuging at 2g on rat long bone metaphyses.

Hypergravity may be considered as a means of counteracting the deleterious effects of microgravity on bone tissue. The effects of exposure to 4 days of hypergravity provided by centrifuging, on bone tissue were studied using histomorphometry. Young 53-day-old male Sprague Dawley rats were randomly divided into a centrifuged group (2g, n = 10), a rotated group (ROTATE, n = 6) of rats exposed to 1.03 g placed in cages near the centre of rotation of the centrifuge and a stationary control group (CONTROL, n = 10). The body mass of the 2g rats was decreased by this experience by 16% compared to CONTROL. The width of the tibial growth plate of 2g was decreased. In two out of ten 2g rats, the hypertrophic zone was injured. In both the tibial and humeral primary (1 degrees ) spongiosae, a reduced 1 degrees spongiosa width (-35% and -24%, ROTATE versus CONTROL respectively; -37% and -41%, 2g versus CONTROL respectively) associated with bone gain (+27% for tibia and humerus ROTATE versus CONTROL; + 16% and +20%, 2g versus CONTROL respectively) was observed in both ROTATE and 2g. In the tibial secondary (2 degrees) spongiosa, bone mass was increased in the 2g (+13% 2g versus CONTROL) rats due to thicker trabeculae, but was decreased in ROTATE rats (-12% versus CONTROL) due to thinner trabeculae. The parameters of formation and resorption activities were stimulated in the 2g and ROTATE groups, the formation activity being more enhanced in 2g. No structural changes were observed in the humeral 2 degrees spongiosa in any of the groups. Numeral bone formation parameters were decreased in 2g and ROTATE but resorption activity was increased in 2g and decreased in ROTATE compared to CONTROL. In conclusion, as early as the 4th day, 2g hypergravity induced reduced endochondral bone formation and increased cancellous bone mass. Rotation led to mixed results including reduced endochondral bone formation, increased bone volume in the 1 degrees spongiosa and bone loss in the 2 degrees spongiosa.

Bisphosphonate effects in rat unloaded hindlimb bone loss model: three-dimensional microcomputed tomographic, histomorphometric, and densitometric analyses.

The effects of antiresorptive drugs on bone loss remain unclear. Using three-dimensional microtomography, dual X-ray/densitometry, and histomorphometry, we evaluated tiludronate effects in the bone loss model of immobilization in tail-suspended rats after 7, 13, and 23 days. Seventy-eight 12-week-old Wistar male rats were assigned to 13 groups: 1 baseline group, and for each time point, 1 control group treated with vehicle and three tail-suspended groups treated with either tiludronate (0.5 or 5 mg/kg) or vehicle, administered s. c. every other day, during the last week before sacrifice. In primary spongiosa (ISP), immobilization-induced bone loss plateaued after day 7 and was prevented by tiludronate. In secondary spongiosa (IISP), bone loss appeared at day 13 with a decrease in trabecular thickness and trabecular number (Tb.N) as assessed by three-dimensional microtomography. Osteoclastic parameters did not differ in tail-suspended rats versus control rats, whereas bone formation showed a biphasic pattern: after a marked decrease at day 7, osteoblastic activity and recruitment normalized at days 13 and 23, respectively. At day 23, the 80% decrease in bone mass was fully prevented by high-dose tiludronate with an increase in Tb.N without preventing trabecular thinning. In summary, at day 7, tiludronate prevented bone loss in ISP. After day 13, tiludronate prevented bone loss in ISP and IISP despite a further decrease in bone formation. Thus, the preventive effects of tiludronate in this model may be related to the alteration in bone modeling with an increase in Tb.N in ISP and subsequently in IISP.

Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three-dimensional contractile collagen gels.

Studies performed at tissular (three-dimensional, 3-D) or cellular (two-dimensional, 2-D) levels showed that the loading pattern plays a crucial role in the osteoblastic physiology. In this study, we attempted to investigate the response of a 3-D osteoblastic culture submitted to either no external stress or static or dynamic stresses. Rat osteosarcoma cells (ROS 17/2.8) were embedded within collagen type I lattices and studied for 3 weeks. Entrapment and proliferation of cells within the hydrated collagen gel resulted in the generation of contractile forces, which led to contraction of the collagen gel. We used this ability to evaluate the influence of three modes of mechanical stresses on the cell proliferation and differentiation: (1) the freely retracted gels (FRG) were floating in the medium, (2) the tense gels (TG) were stretched statically and isometrically, with contraction prevented in the longitudinal axis, and (3) the dynamic gels (DG) were floating gels submitted to periodic stresses (50 or 25 rpm frequency). Gels showed maximum contraction at day 12 in 50 rpm DG, followed by 25 rpm DG, then FRG (88%, 81%, 70%, respectively) and at day 16 in TG (33%). The proliferation rate was greater in TG than in FRG (+52%) but remained low in both DGs. Gel dimensions were related to the collagen concentration and on a minor extent to cell number. Cells in DG appeared rounder and larger than in other conditions. In TG, cells were elongated and oriented primarily along the tension axis. Scanning electron microscopy (SEM) showed that tension exerted by cells in TG led to reorientation of collagen fibers which, in turn, determined the spatial orientation and morphology of the cells. Transmission electron microscopy (TEM) performed at maximum proliferation showed a vast majority of cells with a distended well-developed RER filled with granular material and numerous mitochondria. Alkaline phosphatase activity peaked close to the proliferation peak in FRG, whereas in TG, a biphasic curve was observed with a small peak at day 4 and the main peak at day 16. In DG, this activity was lower than in the two other conditions. A similar time course was observed for alkaline phosphatase gene expression as assessed by Northern blots. Regardless of the conditions, osteocalcin level showed a triphasic pattern: a first increase at day 2, followed by a decrease from day 4 to 14, and a second increase above initial values at day 18. Microanalysis-x indicated that mineralization occurred after 14 days and TEM showed crystals within the matrix. We showed that static and dynamic mechanical stresses, in concert with 3-D collagen matrices, played a significant role on the phenotypic modulation of osteoblast-like cells. This experimental model provided a tool to investigate the significance and the mechanisms of mechanical activity of the 3-D cultured osteoblast-like cells.

Capillary gas chromatographic method for the measurement of small concentrations of monoethylglycinexylidide and lidocaine in plasma.

The major metabolite of lidocaine, monoethylglycinexylidide (MEGX) is currently used as a dynamic marker of liver function. It has been proven, in recent advances, that the determination of MEGX formation after intravenous injection of lidocaine was an effective means of assessing liver dysfunction in critically ill patients. An accurate and sensitive gas chromatographic method has been developed for the determination of small quantities of MEGX formed in such cases. The procedure involves a solid-phase extraction and injection of the extract (splitless mode) into a gas chromatograph equipped with a capillary column and nitrogen-phosphorus detector. The limit of detection is 1 ng/ml and the limit of quantification is 2.5 ng/ml. The response is linear up to 50 ng/ml. The inter- and intra-assay coefficients of variation for MEGX and lidocaine are between 5 and 9%. This method can be used for the determination of small concentrations of MEGX in plasma and could be applied to analysis of small amounts of many other nitrogenous molecules.