Micro‐computed tomographic evaluation of fetal skeletal changes induced by all‐trans‐retinoic acid in rats and rabbits

BACKGROUND: Our laboratory has been conducting positive control studies to evaluate the utility of micro‐computed tomography (micro‐CT) for qualitative evaluation of fetal skeletal morphology. All‐trans‐retinoic acid (atRA) was used to produce a different spectrum of defects compared to our previous studies with boric acid and hydroxyurea. METHODS: Groups of five mated Crl:CD(SD) female rats each were administered vehicle or atRA (2.5–50 mg/kg) on GD 10, and groups of four mated Dutch Belted rabbits each were dosed with vehicle or atRA (6.25–25 mg/kg) on GD 9. Cesarean sections were performed on GD 21 and 28, respectively. Following external examination the viscera were removed and fetuses scanned in a micro‐CT imaging system. Fetuses were subsequently stained with alizarin red. Skeletal morphology was evaluated by each method without the knowledge of treatment group. Total bone mineral content (BMC) of each fetus was quantitated using the micro‐CT images. RESULTS: In rats there were dose‐related increases in the incidence of extra lumbar vertebra and non‐dose‐related increases in supernumerary ribs at all dose levels. There were decreases in mean number of ossified sacrocaudal vertebra at ≥5 mg/kg, and increases in skull bone malformations at ≥10 mg/kg. Rabbits were less sensitive on a mg/kg basis since skeletal malformations and a decrease in mean number of ossified sacrocaudal vertebra were observed only in the 25‐mg/kg group. Micro‐CT evaluation detected essentially the same incidence of skeletal abnormalities as seen in alizarin red‐stained rat and rabbit fetuses. BMC analysis showed a trend toward slight decreases in atRA‐treated rats, but no notable changes in rabbits. CONCLUSIONS: These results add support to our previous work that demonstrates that micro‐CT imaging can effectively assess rat and rabbit fetal skeletal morphology. Birth Defects Res (Part B) 89:408–417, 2010. © 2010 Wiley‐Liss, Inc.     

Relationships between bone mass and micro-architecture at the mandible and iliac bone in edentulous subjects: a dual X-ray absorptiometry, computerised tomography and microcomputed tomography study

doi: 10.1111/j.1741‐2358.2011.00527.x Relationships between bone mass and micro‐architecture at the mandible and iliac bone in edentulous subjects: a dual X‐ray absorptiometry, computerised tomography and microcomputed tomography study Objectives: To compare bone volume, bone mineral density, cortical thickness and bone micro‐architecture in a series of paired mandibular and iliac bone samples analysed by various imagery techniques to see whether relationships exist between the various techniques and between mandibular and iliac bone. Materials and methods: Bone samples from the mandible and ilium were harvested in 20 cadavers and analysed by dual energy X‐ray absorptiometry (DXA), computerised tomography (CT) on a conventional hospital machine and microCT. Results: Significant correlations were found between Hounsfield density obtained by CT, and bone mass determined by microCT but not with DXA values. Cortical thickness measurements were well correlated between CT and microCT. No relationships were found between mandibular and iliac bone, when considering mineral density, cortical thickness, bone volume or micro‐architecture. Conclusion: In clinical practice, CT remains the most appropriate routine means for bone qualitative and quantitative evaluation at the mandible. In this ex vivo study, these results confirm that mandibular bone status does not reflect the axial skeletal one and assist in the placement of implants with dental prostheses in old or osteoporotic patients.     

Development of a 3D Tissue‐Engineered Skeletal Muscle and Bone Co‐culture System

In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.     

X‐ray microtomography (micro‐CT): a reference technology for high‐resolution quantification of xylem embolism in trees

As current methods for measuring xylem embolism in trees are indirect and prone to artefacts, there is an ongoing controversy over the capacity of trees to resist or recover from embolism. The debate will not end until we get direct visualization of the vessel content. Here, we propose desktop X‐ray microtomography (micro‐CT) as a reference direct technique to quantify xylem embolism and thus validate more widespread measurements based upon either hydraulic or acoustic methods. We used desktop micro‐CT to measure embolism levels in dehydrated or centrifuged shoots of laurel – a long‐vesseled species thought to display daily cycles of embolism formation and refilling. Our direct observations demonstrate that this Mediterranean species is highly resistant to embolism and is not vulnerable to drought‐induced embolism in a normal range of xylem tensions. We therefore recommend that embolism studies in long‐vesseled species should be validated by direct methods such as micro‐CT to clear up any misunderstandings on their physiology.     

Teratogenicity of zinc deficiency in the rat: study of the fetal skeleton

Zinc deficiency (ZD) is teratogenic in rats, and fetal skeletal defects are prominent. This study identifies fetal skeletal malformations that affect calcified and non-calcified bone tissue as a result of gestational zinc deficiency in rats, and it assesses the effect of maternal ZD in fetal bone calcification. Pregnant Sprague-Dawley rats (180-250 g) were fed 1) a control diet (76.4 micrograms Zn/g diet) ad libitum (group C), 2) a zinc-deficient diet (0 microgram/g) ad libitum (group ZD), or 3) the control diet pair-fed to the ZD rats (group PF). On day 21 of gestation, laparotomies were performed. Fetuses were weighed, examined for external malformations, and stained in toto with a double-staining technique for the study of skeletal malformations. Maternal and fetal tissues were used for Zn, Mg, Ca, and P determinations. Gross external malformations were present in 97% of the ZD fetuses. No external malformations were found in fetuses from groups C and PF. Ninety-one percent of cleared ZD fetuses had multiple skeletal malformations, whereas only 3% of the fetuses of group PF had skeletal defects; no skeletal malformations were found in fetuses from group C. Some of the skeletal malformations described in the ZD fetuses, mainly affecting non-calcified bone, were not mentioned in previous reports, thus stressing the importance of using double-staining techniques. Examination of stained fetuses and counting of ossification centers revealed important calcification defects in ZD fetuses. These effects were confirmed by lower Ca and P concentrations in fetal bone with alteration of the Ca:P ratio.     

A new high‐resolution computed tomography (CT) segmentation method for trabecular bone architectural analysis

In the last decade, high‐resolution computed tomography (CT) and microcomputed tomography (micro‐CT) have been increasingly used in anthropological studies and as a complement to traditional histological techniques. This is due in large part to the ability of CT techniques to nondestructively extract three‐dimensional representations of bone structures. Despite prior studies employing CT techniques, no completely reliable method of bone segmentation has been established. Accurate preprocessing of digital data is crucial for measurement accuracy, especially when subtle structures such as trabecular bone are investigated. The research presented here is a new, reproducible, accurate, and fully automated computerized segmentation method for high‐resolution CT datasets of fossil and recent cancellous bone: the Ray Casting Algorithm (RCA). We compare this technique with commonly used methods of image thresholding (i.e., the half‐maximum height protocol and the automatic, adaptive iterative thresholding procedure). While the quality of the input images is crucial for conventional image segmentation, the RCA method is robust regarding the signal to noise ratio, beam hardening, ring artifacts, and blurriness. Tests with data of extant and fossil material demonstrate the superior quality of RCA compared with conventional thresholding procedures, and emphasize the need for careful consideration of optimal CT scanning parameters. Am J Phys Anthropol 2009. © 2009 Wiley‐Liss, Inc.     

Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs

Whole‐organ and whole‐body optical tissue clearing methods allowing imaging in 3 dimensions are an area of profound research interest. Originally developed to study nervous tissue, they have been successfully applied to all murine organs, yet clearing and imaging of rat peripheral organs is less advanced. Here, a modification of CUBIC clearing protocol is presented. It provides a rapid and simple approach to clear the entire adult rat organism and thus all organs within as little as 4 days. Upgraded perfusion‐based rat CUBIC protocol preserves both anatomical structure of organs and signal from proteinaceous fluorophores, and furthermore is compatible with antibody staining. Finally, it enables also volumetric cells analyses and is tailored for staining of calcium deposits within unsectioned soft tissues.      

Artificial selection sheds light on developmental mechanisms of limb elongation

Species diversity in limb lengths and proportions is thought to have evolved adaptively in the context of locomotor and habitat specialization, but the heritable cellular processes that drove this evolution within species are poorly understood. In this study, we take a novel “micro‐evo‐devo” approach, using artificial selection on relative limb length to amplify phenotypic variation in a population of mice, known as Longshanks, to examine the cellular mechanisms of postnatal limb development that contribute to intraspecific limb length variation. Cross‐sectional growth data indicate that differences in bone length between Longshanks and random‐bred controls are not due to prolonged growth, but to accelerated growth rates. Histomorphometric and cell proliferation assays on proximal tibial growth plates show that Longshanks’ increased limb bone length is associated with an increased number of proliferative chondrocytes. In contrast, we find no differences in other growth plate cellular features known to underlie interspecific differences in limb bone size and shape, such as the rates of chondrocyte proliferation or the size and number of hypertrophic cells in the growth plate. These data suggest that small differences among individuals in the number of proliferating chondrocytes are a potentially important determinant of selectable intraspecific variation in individual limb bone lengths, independent of body size.     

The Potential of Cr3 [Triaqua‐μ3‐Oxo‐Hexa‐μ‐Propionatotrichromium(III) Chloride] to Reduce Birth Defects in the Offspring of Diabetic CD‐1 Mice

Diabetes mellitus is a growing concern worldwide and leads to multiple complications during pregnancy. Pharmacologic doses of chromium (Cr) have been linked with improving insulin sensitivity and other positive benefits in the treatment of diabetes in animal models. By using streptozotocin induced hyperglycemia in female CD‐1 mice, reproductive outcomes of diabetic and chromium‐dosed diabetic females were examined. After dosing 10 mg/kg Cr in the form of triaqua‐μ₃‐oxo‐hexa‐μ‐propionatotrichromium(III) chloride or Cr3 during gestation days 8–16 (GD8–GD16), all females were sacrificed on gestation day 17 (GD17) and examined for maternal weight gain. The fetuses were examined for gross malformations and for skeletal malformations. The offspring of Cr3‐dosed females tended to have a reduction in the incidence of supernumerary ribs. While hyperglycemia still had negative impacts on the health of dams and their offspring, administration of Cr led to an apparent trend in the reduction in the number of malformations and incidence of supernumerary ribs compared to those of untreated diabetic mothers     

Whole‐field macro‐ and micro‐deformation characteristic of unbound water‐loss in dentin hard tissue

High‐resolution deformation measurements in a functionally graded hard tissue such as human dentin are essential to understand the unbound water‐loss mediated changes and their role in its mechanical integrity. Yet a whole‐field, 3‐dimensional (3D) measurement and characterization of fully hydrated dentin in both macro‐ and micro‐scales remain to be a challenge. This study was conducted in 2 stages. In stage‐1, a stereo‐digital image correlation approach was utilized to determine the water‐loss and load‐induced 3D deformations of teeth in a sagittal section over consecutively acquired frames, from a fully hydrated state to nonhydrated conditions for a period up to 2 hours. The macroscale analysis revealed concentrated residual deformations at the dentin‐enamel‐junction and the apical regions of root in the direction perpendicular to the dentinal tubules. Significant difference in the localized deformation characteristics was observed between the inner and outer aspects of the root dentin. During quasi‐static loadings, further increase in the residual deformation was observed in the dentin. In stage‐2, dentin microstructural variations induced by dynamic water‐loss were assessed with environmental scanning electron microscopy and atomic force microscopy (AFM), showing that the dynamic water‐loss induced distention of dentinal tubules with concave tubular edges, and concurrent contraction of intertubular dentin with convex profile. The findings from the current macro‐ and micro‐scale analysis provided insight on the free‐water‐loss induced regional deformations and ultrastructural changes in human dentin.      

Toxicity of bryostatin‐1 on the embryo‐fetal development of Sprague‐Dawley rats

BACKGROUND: Bryostatin‐1, a highly oxygenated marine macrolide with a unique polyacetate backbone isolated from the marine animal Bugula neritina (Linnaeus), is now being developed as an anti‐cancer drug for treating malignancy. In the present study, developmental toxicity of bryostatin‐1 was evaluated in Sprague–Dawley rats. METHODS: Bryostatin‐1 was intravenously administered to rats on gestation days 6–15 at 4.0, 8.0, and 16.0 µg/kg on a daily basis. Then the reproductive parameters were determined in animals, and fetuses were examined for external, visceral, and skeletal malformations. RESULTS: The total weight gains were significantly different in animals between the control group and 8.0 and 16.0 µg/kg bryostatin‐1 groups during and after treatment. The resorption and death fetus rates were significantly different between the bryostatin‐1 group (16 µg/kg) and the control group. The fetal weight and fetal crown‐rump length in the bryostatin‐1 groups were significantly lower than that in the control group. CONCLUSIONS: Our results indicated that maternal toxicity occurred when the dose of bryostatin‐1 was at 8.0 µg/kg, embryotoxicity at 16.0 µg/kg, and fetotoxicity at 4.0 µg/kg; but bryostatin‐1 showed no teratogenic effect in rats. In light of our findings, bryostatin‐1 should be used with caution in pregnant women with cancer, if they would like to continue the pregnancy. Birth Defects Res (Part B) 89:171–174, 2010. © 2010 Wiley‐Liss, Inc.     

MiR‐106b inhibition suppresses inflammatory bone destruction of wear debris‐induced periprosthetic osteolysis in rats

Aseptic loosening caused by periprosthetic osteolysis (PPO) is the main reason for the primary artificial joint replacement. Inhibition of inflammatory osteolysis has become the main target of drug therapy for prosthesis loosening. MiR‐106b is a newly discovered miRNA that plays an important role in tumour biology, inflammation and the regulation of bone mass. In this study, we analysed the in vivo effect of miR‐106b on wear debris‐induced PPO. A rat implant loosening model was established. The rats were then administrated a lentivirus‐mediated miR‐106b inhibitor, miR‐106b mimics or an equivalent volume of PBS by tail vein injection. The expression levels of miR‐106b were analysed by real‐time PCR. Morphological changes in the distal femurs were assessed via micro‐CT and histopathological analysis, and cytokine expression levels were examined via immunohistochemical staining and ELISA. The results showed that treatment with the miR‐106b inhibitor markedly suppressed the expression of miR‐106b in distal femur and alleviated titanium particle‐induced osteolysis and bone loss. Moreover, the miR‐106b inhibitor decreased TRAP‐positive cell numbers and suppressed osteoclast formation, in addition to promoting the activity of osteoblasts and increasing bone formation. MiR‐106b inhibition also significantly regulated macrophage polarization and decreased the inflammatory response as compared to the control group. Furthermore, miR‐106b inhibition blocked the activation of the PTEN/PI3K/AKT and NF‐κB signalling pathways. Our findings indicated that miR‐106b inhibition suppresses wear particles‐induced osteolysis and bone destruction and thus may serve as a potential therapy for PPO and aseptic loosening.     

Bone quality in prehistoric,cis‐baikal forager femora: A micro‐CT analysis of cortical canal microstructure

Bone quality, a contributor to bone strength, is determined by structural and mechanical properties, which may be analyzed by gross and/or microscopic methods. Variables that contribute to bone quality, such as porosity, can provide insight into the health and lifestyles of people in prehistory. This study tests the ability of microcomputed tomography (µCT) to capture and characterize cortical canal systems in archaeological bone. Seven variables and 71 femora are analyzed to explore bone dynamics in prehistoric foragers from Lake Baikal, Siberia. The results indicate that canal number and canal separation differ significantly (P < 0.05) between age‐at‐death categories, but only for the pooled and male samples. When merged into a new variable by means of principal components analysis, canal diameter and canal surface to canal volume are also able to discriminate amongst age‐at‐death categories, as well as between the sexes. However, the overall lack of significant differences between the sexes and amongst age‐at‐death categories indicates that Baikal forager bone quality (i.e., canal architecture) did not change drastically throughout the lifespan. Interestingly, principal component one identified an untested variable that contributes to canal microstructure variability, and a sexual division of labor may promote divergent trends in canal degree of anisotropy between the sexes. Overall, µCT provides an alternate method for exploring bone quality in archaeological remains, complementing existing methods such as thin‐sectioning and gross morphological analyses. Am J Phys Anthropol 154:486–497, 2014. © 2014 Wiley Periodicals, Inc.     

Novel micro‐bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed‐batch CHO cultures

With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench‐top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high‐throughput (HT) technology for process development. One such high‐throughput system is the SimCell? platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell? micro‐bioreactor technology for fed‐batch cultivation of a GS‐CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas‐permeable chambers based on a micro‐fluidic design, with six micro‐bioreactors (MBs) per micro‐bioreactor array (MBA). Online, non‐invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3‐ and 100‐L bioreactor scales. The results of the study demonstrate that the SimCell? platform operated under fed‐batch conditions could support viable cell concentrations up to least 12 × 10⁶ cells/mL. In addition, both intra‐MB (MB to MB) as well as intra‐MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra‐MB and ‐MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) × 100. The % CV values for most intra‐MB and intra‐MBA measurements were generally under 10% and the intra‐MBA values were slightly lower than those for intra‐MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench‐top, and pilot scale bioreactor cultivations and found to be within ±20% of the historical averages. Biotechnol. Bioeng. 2010; 106: 57–67. © 2010 Wiley Periodicals, Inc.     

Licorice extract increases cyclophosphamide teratogenicity by upregulating the expression of cytochrome P-450 2B mRNA

BACKGROUND: Since cyclophosphamide is metabolically activated to teratogenic acrolein and cytotoxic phosphoramide mustard by cytochrome P‐450 type 2B (CYP2B), we assessed the effects of licorice, a CYP2B inducer, on the fetal defects induced by cyclophosphamide. METHODS: Pregnant Sprague‐Dawley rats were daily administered with licorice (100 mg/kg) by gavage for 7 days, from the 6th to 12th day of gestation, and intraperitoneally administered with cyclophosphamide (11 mg/kg) 1 hr after the final licorice treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarian section. RESULTS: Cyclophosphamide was found to reduce fetal and placental weights without increasing resorption or death. In addition, it induced malformations in live fetuses; 93.8, 41.1, and 100% of the external (skull and limb defects), visceral (cleft palate and ureteric dilatation), and skeletal (acrania, vertebral/costal malformations, and delayed ossification) abnormalities, respectively. When pre‐treated with licorice, cyclophosphamide‐induced body weight loss and abnormalities of fetuses were remarkably aggravated. Moreover, repeated treatment with licorice greatly increased mRNA expression and activity of hepatic CYP2B. CONCLUSIONS: The results indicate that repeated intake of licorice may aggravate cyclophosphamide‐induced body weight loss and malformations of fetuses by upregulating CYP2B. Birth Defects Res (Part B) 92:553–559, 2011. © 2011 Wiley Periodicals, Inc.     

2‐D PAGE and MS analysis of proteins from formalin‐fixed,paraffin‐embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.     

High-throughput staining for the evaluation of fetal skeletal development in rats and rabbits

Typical developmental toxicity studies require the assessment of fetal skeletal development. Regulatory guidelines require the assessment of bone ossification and indicate preferences for an assessment of both ossified bone as well as cartilaginous elements. Current manual methods to process fetuses for skeletal examination, whether single or double staining, are laborious and time consuming, and ultimately extend the time before study interpretations. There is a definite need for a quick and efficient, yet reliable, procedure to generate stained fetal skeletons for analysis. A non-automated high-throughput method for single and double staining rat and rabbit fetuses for skeletal evaluations is described, which results in excellent quality specimens ready for evaluations in approximately 3 days for rats and 7 days for rabbits.