A new aid to aging immature skeletons: development of the occipital bone

A total of 150 immature and young adult skeletons from the medieval necropolis at Mistihalj, in southern Yugoslavia, were aged by the standard methods: dentition, osseous development, and extent of vertebral union. The development of the occipital bone was examined in 117 cases to determine if it could be used in aging the skeletons. The stages of development were found to be regular enough to permit reasonable prediction of age for other Yugoslavian specimens. Comparisons with a series of 20 German fetuses suggest measurements of the parts of the occipital bone can be used to help determine the approximate age of skeletons in the fetal to few-months post-natal range. Comparisons with the ten Egyptian skulls and data on 19 unpublished Eskimo skeletons suggest that the stages of development can be used with other populations.     

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.     

An automated technique for double staining mouse fetal and neonatal skeletal specimens to differentiate bone and cartilage

Historically, some fetuses for regulatory developmental toxicity studies have been stained with alizarin red S and cleared with glycerol to visualize the ossified portion of their skeletons. Interest in examining cartilage arose owing to its inclusion in some regulatory guidelines. Methods for double staining rat skeletons have been published previously. The method described here for staining mouse skeletons is fully automated and uses alizarin red S to stain bone and Alcian blue to stain cartilage. Pregnant mice (Crl:CD1) were euthanized on gestation day 18 to obtain fetal specimens. Day 0 post-partum mouse pups also were stained. Our method was developed using the Shandon Pathcentre , which is a fully enclosed automated staining system that allows staining to be carried out at 30° C with a final clearing at 35° C. Our method uses the same solutions as for fetal rat processing, but with reduced time periods for the smaller size of mice vs. rat specimens. Staining, maceration and clearing of the specimens requires approximately 2 days. The time required of laboratory personnel, however, is minimal, because all solutions are changed automatically and the specimens do not require examination or removal from the processor until processing is complete. After processing, the specimens are suitable for immediate assessment of bone and cartilage. A mouse developmental toxicity study using 20 animals/group and approximately 10 fetuses/animal could be processed in only three runs using one machine.     

Age and sex diagnosis of the skeleton of immature individuals

In connection with increasing theory-bound investigations in prehistoric anthropology age and sex diagnosis of children's skeletons is gaining importance. Developmental stages of the tympanic plate are introduced as an additional criterion for ageing infants. Discriminant functions derived from ilium and femur measurements allow preliminary access towards a metric sexual diagnosis of fetal and neonate individuals.     

Large-scale double-staining of rat fetal skeletons using Alizarin Red S and alcian blue

A critical component in the conduct of a prenatal developmental toxicity study is the evaluation of fetal skeletal development. As the developing rodent fetus is typically evaluated at gestation day 20, at a time when ossification of the skeleton is incomplete, a thorough assessment of skeletal development would include both ossified and cartilaginous structures. Current methods to double-stain the fetal skeleton using Alizarin Red S and Alcian Blue are typically described for small sample sizes or using time allotments for each processing step that are unsuitable for industry. In an industrial setting, there is a need for an effective means to double-stain fetal skeletons on a large scale (i.e., hundreds of fetuses simultaneously). This article describes a method used in our laboratory to stain both fetal bone and cartilage using solutions and procedures on an industrial scale.     

The density of long limb bones and the percentage ash weight of the skeleton of Macaca mulatta

The gravimetric density of humeri, radii, femora and tibiae from a series of 274 male and female skeletons of rhesus monkeys, Macaca mulatta, was determined for fetal, young and adult periods. The ages of 171 of the animals were known: they ranged from 57 days of gestation to 13.6 years; the ages of an additional 103 skeletons were estimated. The mean density of the fetal bones was found to increase linearly with age and was higher for males than females, and higher for the superior than for the inferior limb bones. During the young period the pattern of increase in density can be represented by a power-type curve, and the density is significantly higher in females than in males and in superior than in inferior limb bones. The densities of the long limb bones of the adult skeletons show a slight, but not significant, negative trend with increasing age. In this age group the mean densities are higher for males than females and higher for the superior than for the inferior limb bones. The percentage ash weight was determined for the total skeleton and for 21 subdivisions of 23 postnatal skeletons with estimated ages. The skull and long limb bones were found to have higher mean percentage ash weights than the vertebral segments and the sternum. Both the density and the percentage ash weight of the Macaca mulatta skeletons examined exceed those found in our earlier studies of the human skeleton.     

Long bone lengths and gestational age distributions of post-contact period Arikara Indian perinatal infant skeletons

Prenatal growth is adversely affected by poor socioeconomic conditions where disease and chronic undernutrition prevail. Premature and small-for-gestational-age births occur at higher frequency. Post-contact Arikara Indian populations of South Dakota experienced a rapidly changing and disruptive environment that included deterioration of the subsistence base and increased morbidity. This research tests resulting fetal growth effects through comparative analysis of two perinatal infant samples of the early (A.D. 1600-1733) and the late (A.D. 1760-1835) post-contact period. Perinatal infants recovered from late cemeteries include a higher percentage of smaller skeletons, as determined using long bone diaphyseal lengths, than is documented for the earlier time period. This contrast shows that it is possible to detect fetal growth differences in samples of archaeological context.     

Skeletal structures and habitats of Recent and fossil Acanthochaetetes (subclass Tetractinomorpha,Demospongiae, Porifera)

The investigation of the habitats, the spicular skeletons, and the structure and chemistry of the nonspicular high-Mg calcite skeletons of a fossil Acanthochaetetes from the Late Albian (Cretaceous) of Northern Spain and the extant Acanthochaetetes wellsi from Pacific reefs demonstrates an astonishing correspondence. The skeletons of both species are hemispherical or pyriform with the lower part containing an epitheca. They are built up of single calicles which are subdivided by tabulae. Spines protrude from the walls into the calicles. Scanning electron microscopy and thin sections reveal that the high-Mg calcite skeleton consists of two different microstructures: a irregular ssensu Wendt 1979 or microlamellar (sensu Cuif et al. 1979) and a completely irregular structure. AAS and EDAX analysis of the calcite skeletons produce roughly the same Mg and Sr contents. Tylostyle megascleres and aster-like microscleres are observed in the spicular skeletons of both species. The only difference between the two species is the greater variability of the microscleres in the extant species. Moreover, the fossil species incorporates the scleres in the non-spicular skeleton, while the extant species does not. Both species live/lived in the same niches of tropical reefs: the cryptic habitats of submarine caves in the reef core and the dimly lighted habitats of the deeper fore-reef.     

The influence of moderate exercise in diabetic and normal pregnancy on maternal and fetal outcome in the rat

The effect of treadmill exercise prior to and during pregnancy on maternal and fetal outcome was studied in nondiabetic and streptozotocin-induced diabetic rats. Animals were exercised daily on a motorized treadmill (16.1 m/min, 45 min/d) for three weeks prior to mating and throughout gestation. The catabolic state of diabetes was evidenced by changes in maternal body composition. Overall, fetuses of diabetic dams were smaller, lighter, had less calcified skeletons and had more malformations compared to control fetuses. Exercise in the nondiabetic dams resulted in a retardation of skeletal ossification compared to fetuses from sedentary controls. However, exercise improved fetal outcome in diabetic rats, resulting in increased fetal weight and a lower frequency of malformations compared to fetuses from sedentary diabetic dams.     

The influence of fibroblast-like cells derived from canine fetal hematopoietic tissues on the regulation of lymphohematopoiesis

Media conditioned by fibroblast-like cells derived from organs active in fetal lymphohematopoiesis were studied for their effects on adult granulocyte/macrophage colony-forming units (CFU-GM). Fibroblasts from fetal liver produced a factor stimulatory for CFU-GM, whereas fibroblasts from fetal marrow produced a factor inhibitory for CFU-GM which was not completely relieved by adding indomethacin to the assay. Our studies indicated that neither fetal marrow nor fetal liver produced factors affecting lymphocyte colony-forming units (CFU-L). Cell-cell interactions between fibroblast-like cells derived from fetal liver or marrow and normal adult CFU-GM were also studied. We observed that fibroblasts derived from both fetal and adult marrow inhibited colony formation, whereas inhibition in the presence of fetal liver fibroblasts was minimal. Loss of inhibitory activity by a liver fibroblast cell line over repeated passages was seen. Differential analysis of colonies formed above an adherent layer of fetal marrow fibroblasts suggested that these fibroblasts suppress myeloid/macrophage differentiation to a far greater degree than did adult marrow fibroblasts. A role in the regulation of fetal lymphohematopoiesis may be played by stromal fibroblasts.     

A composite transposon 3'' to the cow fetal globin gene binds a sequence specific factor.

Two unusual sequence organizations were found within the beta-globin locus of the cow. Each was a composite, consisting of closely linked Alu-type repeats with a short stretch of genomic non-repetitive sequence, called a lagan, sandwiched between. One lagan was found 3' to the fetal globin gene, while the second lay between the adult globin gene and a globin pseudogene. Southern blot analysis indicated that both lagans appeared twice within the cow haploid genome, with the second copies lying outside the cow beta-globin locus. One of these non-globin locus homologues was cloned and subjected to sequence analysis. Comparison of the DNA sequence data showed that the lagan-Alu composite was transposed as a unit. The lagan 3' to the cow fetal globin gene contains the recognition site for a sequence specific DNA binding factor. This factor was present in extracts from fetal, but not from adult cow tissues.     

Ancient skeletal remains of the Canary Islands: bone histology and chemical analysis.

Prehispanic burials from the Canary Islands are often well preserved. Many of the bodies are mummified, most of them were not interred, but deposited in caves. Bone histological and trace element analysis of 117 skeletons of the prehispanic period of the Canary Islands was performed. In some of the islands we have found a high prevalence of osteoporosis, whereas in others, histomorphometrically assessed trabecular bone mass (TBM) (in undecalcified iliac crest specimens) was in the normal range. Bone trace elements analysis have shown high bone S(r), Mg and Mn, and low Fe, Zn and Cu in those skeletons with a more reduced TBM. These facts speak for a relative protein-calorie malnutrition and a consumption of a mainly vegetarian diet. This is especially marked in the skeletons from Gran Canaria.     

The apical and basal plasma membranes of the human placental syncytiotrophoblast contain different erythrocyte membrane protein isoforms. Evidence for placental forms of band 3 and spectrin

Using immunochemical techniques, we identified forms of erythrocyte membrane proteins in apical and basal plasma membranes of human placental trophoblast. A wheat germ agglutinin-binding intrinsic protein was present in the microvillous (maternal facing) but not the basal (fetal facing) membrane of the syncytiotrophoblast epithelium. Conversely, erythrocyte-related proteins of the basal membrane included two intrinsic membrane proteins, a 95,000 Mr band 3 isoform and a form of spectrin. These four proteins were all absent from the microvillous membrane. The basal membrane spectrin isoform was also present in basal membrane skeletons. A 70,000 Mr polypeptide which reacted with antibodies to band 3 was present in both microvillous and basal plasma membranes. Therefore, certain isoforms of red cell membrane proteins are polarized between the two surfaces of the human placental syncytiotrophoblast. We propose that the localization of spectrin to the basal membrane is related to the less bundled organization of microfilaments at this membrane compared with that of the microvillous membrane. The band 3 isoforms are candidates for participation in maternofetal anion transport.     

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear‐staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro‐CT) imaging is a versatile, well‐documented tool traditionally used to generate high‐resolution 3‐D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro‐CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro‐CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro‐CT measurement of fetal limb lengths correlates well with traditional clear‐staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro‐CT and 10.82% for clear‐staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro‐CT technique to generate 3‐D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear‐staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well. Birth Defects Res (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

Sex differences in the Ilia of a known sex and age sample of fetal and infant Skeletons

Ilia from a known sex, race, and age sample of fetal and infant skeletons from the collections of the Smithsonian Institution were evaluated for six metric and one nonmetric characters. Three indices calculated from the measurements were analyzed. The nonmetric trait was examined for fit with known sex. The three indices failed to show significant sex differences. The nonmetric trait, Auricular Surface Elevation, proved dependable (91% accurate) for the male Fetal and Six Month age groups.     

Age determination of pre- and postnatal skeletons with special reference to current methodological aspects]

From the remains of ten fetal and neonatal skeletons the standard methods of age estimation by means of dental and osseous criteria as well as diaphyseal length were applied and discussed. The ratio of long bone lengths and change of proportions during the first and second half of pregnancy is a useful indicator for the stage of maturation.     

Occurrence and diversity of lipids in modern coral skeletons

Coral skeletons are composite acellular structures, in which organic macromolecules are intimately associated with mineral phases. Previous studies focussed on proteins and sugars of the soluble organic matrices extracted from the skeletons. Here we report the occurrence of diverse lipids which were extracted from the aragonitic skeletons of seven modern coral species. Using thin layer chromatography, we show that these lipids differ in quantity and composition between the species. Higher proportions of sterols and sterol esters in skeleton extracts as compared to a much higher abundance of waxes and triglycerides in previously studied extracts from scleractinian soft tissues suggest a specific, although not yet determined, role in biomineralisation. The occurrence of intraskeletal lipids along with other organic components should also be taken into account when using coral skeletons as bone allografts, as well as in fossilisation processes.     

Conformation and elasticity of the isolated red blood cell membrane skeleton.

We studied the structure and elasticity of membrane skeletons from human red blood cells (RBCs) during and after extraction of RBC ghosts with nonionic detergent. Optical tweezers were used to suspend individual cells inside a flow chamber, away from all surfaces; this procedure allowed complete exchange of medium while the low-contrast protein network of the skeleton was observed by high resolution, video-enhanced differential interference-contrast (DIC) microscopy. Immediately following extraction in a 5 mM salt buffer, skeletons assumed expanded, nearly spherical shapes that were uncorrelated with the shapes of their parent RBCs. Judging by the extent of thermal undulations and by their deformability in small flow fields, the bending rigidity of skeletons was markedly lower than that of either RBCs or ghosts. No further changes were apparent in skeletons maintained in this buffer for up to 40 min at low temperatures (T less than 10 degrees C), but skeletons shrank when the ionic strength of the buffer was increased. When the salt concentration was raised to 1.5 M, shrinkage remained reversible for approximately 1 min but thereafter became irreversible. When maintained in 1.5 M salt buffer for longer periods, skeletons continued to shrink, lost flexibility, and assumed irregular shapes: this rigidification was irreversible. At this stage, skeletons closely resembled those isolated in standard bulk preparations. We propose that the transformation to the rigid, irreversibly shrunken state is a consequence of spectrin dimer-dimer reconnections and that these structural rearrangements are thermally activated. We also measured the salt-dependent size of fresh and bulk extracted skeletons. Our measurements suggest that, in situ, the spectrin tethers are flexible, with a persistence length of approximately 10 nm at 150 mM salt.     

Similarities in structure and function of bovine serum erythrotropin and human insulin-like growth factor II: two fetal erythroid cell stimulating factors

Serum erythrotropin (ET) was isolated from fetal bovine serum. Partial sequence analysis of the N-terminal portion of the peptide indicated that the first 20 amino acids were practically identical to those found in human insulin-like growth factor II (IGF II). The effect of IGF II on [3H] thymidine incorporation in cell cultures of fetal bovine liver was similar to the effect of ET. Both factors acted synergistically with erythropoietin but not with platelet derived growth factor. The stimulation of thymidine incorporation by ET and IGF II on cell cultures of fetal liver erythroid cells was at least 15 times higher than their effects on cultures of fetal calf intestine, lung and kidney cells.     

Cell spreading factor. Occurrence and specificity of action.

A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.