Differences in fetal fractional limb volume changes in normal and gestational diabetic pregnancies: an exploratory observational study.

OBJECTIVE: Fetal fractional limb volume has been proposed as a useful measure for quantifying fetal soft tissue development. The aim of this study was to investigate the growth of fractional arm volume (AVol) and fractional thigh volume (TVol) of fetuses with maternal gestational diabetes (GDM) compared with those of fetuses with normal glucose tolerance (NGT). We hypothesised fetal fractional limb volume would be larger in the GDM group than in the NGT group in late gestation. DESIGN: Exploratory observational study. SETTING: Saitama Municipal Hospital. SAMPLE: A total of 165 (125 NGT and 40 GDM) singleton Japanese pregnant women. METHODS: AVol and TVol were assessed between 20 and 37 weeks' gestation as cylindrical limb volumes based on 50% of the fetal humeral or femoral diaphysis length. Women were diagnosed as GDM based on the criteria of the Japan Society of Obstetrics and Gynecology. MAIN OUTCOME MEASURES: AVol and TVol were compared between women with NGT and those with GDM at each gestational age period (2-week intervals from 20 to 37 weeks' gestation). RESULTS: Overall, 287 ultrasound scans were performed (NGT group, 205 scans; GDM group, 82 scans). There was no significant difference of AVol between the groups before 32 weeks' gestation. AVol was significantly larger in the GDM group than in the NGT group after 32 weeks' gestation (P < 0.05). TVol was not statistically different between the groups across gestation. CONCLUSIONS: Detection of variations in fetal AVol may provide greater insight into understanding the origins of altered fetal body proportion in GDM. TWEETABLE ABSTRACT: AVol, but not TVol, is significantly larger in fetuses with GDM than in those with NGT after 32 weeks' gestation.

Changes in Proximal Femoral Shape During Fetal Development.

BACKGROUND: Walker and Goldsmith's classic article on fetal hip joint development reported that neck/shaft angle did not change from 12 weeks of gestational age through term while version increased from 0 to 40 degrees. This suggests no change in coronal alignment during development, a conclusion we dispute. By re-examining their data, we found that the true neck/shaft angle (tNSA) decreased by 7.5 degrees as version increased by 40 degrees from 12 weeks of gestational age to term. METHODS: Four investigators measured both femoral version and neck-shaft angle from photographs published by the authors of femurs at multiple stages of maturation from 12 weeks of gestational age to term. The tNSAs and inclination angles were calculated for each femur illustrated using previously validated formula. Changes in the morphology of the femur over time were analyzed using a Student t test. Interobserver and intraobserver reliability were also determined by the Pearson R coefficient. RESULTS: As reported by Walker and Goldsmith, apparent neck/shaft angle (aNSA) did not significantly change during maturation, whereas version increased by 40 degrees. However, tNSA decreased by 7.5 degrees during maturation, while the inclination increased by 32 degrees over the same period. This paper demonstrates angular changes in both the coronal and transverse planes with a 4:1 ratio of angular change in the transverse and coronal planes respectively. Interobserver Pearson coefficient R=0.98 and an intraobserver Pearson coefficient R=0.99. CONCLUSIONS: Although Walker and Goldsmith reported angular changes only in the transverse plane, we conclude that they identified angular changes in both the coronal and transverse planes. Here we show it is mathematically necessary for tNSA to decrease, if aNSA remains constant as version increases. CLINICAL RELEVANCE: A reader who is not well versed in the difference between aNSA and tNSA or version and inclination cannot appreciate what Walker and Goldsmith presented. Surgeons operating on the proximal femur also benefit from understanding these distinctions.

Widening of the femoral proximal diaphysis--metaphysis angle in fetuses with achondroplasia.

OBJECTIVES: It has recently been reported that fetuses with achondroplasia have a wider than expected femoral proximal diaphysis-metaphysis angle (femoral angle). The aim of this case-control study was to investigate this finding. METHODS: Cases with confirmed achondroplasia (n = 6), small-for-gestational-age fetuses (n = 70) and a group of normal fetuses (n = 377) were included in this study. The ultrasound image of the femur was examined by two independent experienced observers blinded to the diagnosis, who measured the femoral angle. These values were converted into multiples of the expected median (MoM), after adjustment for gestational age and femur length. Prevalence of various prenatal ultrasound signs of achondroplasia was determined in affected fetuses. Intra- and interobserver agreement of measurement of femoral angle was assessed using 95% limits of agreement and kappa statistics. RESULTS: The femoral angle can be measured accurately by ultrasound, and increases with both increasing gestational age and increasing femur length. The femoral angle-MoM was significantly higher in fetuses with achondroplasia than in the control group (1.36 vs 1.00 MoM, P < 0.001) and in the SGA group (1.36 vs 1.04 MoM, P < 0.001). It measured more than 130° in five of the six cases with achondroplasia (83.3%), which was the most consistent finding other than shortening of the long bones. CONCLUSIONS: The femoral angle is wider in fetuses with achondroplasia. This new ultrasound sign appears promising as an additional discriminatory marker when clinicians are faced with a case of short long bones in the third trimester.

Development of the tarsometatarsal skeleton by the lateral fusion of three cylindrical periosteal bones in the chick embryo (Gallus gallus).

An avian tarsometatarsal (TMT) skeleton spanning from the base of toes to the intertarsal joint is a compound bone developed by elongation and lateral fusion of three cylindrical periosteal bones. Ontogenetic development of the TMT skeleton is likely to recapitulate the changes occurred during evolution but so far has received less attention. In this study, its development has been examined morphologically and histologically in the chick, Gallus gallus. Three metatarsal cartilage rods radiating distally earlier in development became aligned parallel to each other by embryonic day 8 (ED8). Calcification initiated at ED8 in the midshaft of cartilage propagated cylindrically along its surface. Coordinated radial growth by fabricating bony struts and trabeculae resulted in the formation of three independent bone cylinders, which further became closely apposed with each other by ED13 when the periosteum began to fuse in a back-to-back orientation. Bone microstructure, especially orientation of intertrabecular channels in which blood vasculature resides, appeared related to the observed rapid longitudinal growth. Differential radial growth was considered to delineate eventual surface configurations of a compound TMT bone, but its morphogenesis preceded the fusion of bone cylinders. Bony trabeculae connecting adjacent cylinders emerged first at ED17 in the dorsal and ventral quarters of intervening tissue at the mid-diaphyseal level. Posthatch TMT skeleton had a seemingly uniform mid-diaphysis, although the septa persisted between original marrow cavities. These findings provide morphological and histological bases for further cellular and molecular studies on this developmental process.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects bone tissue in rhesus monkeys.

Bone tissue is one of the target tissues for dioxins and dioxin-like compounds. Therefore, the aim of this study was to investigate effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on bone tissue in rhesus monkey, the most human-like experimental model available. Pregnant rhesus monkeys (Macaca mulatta; age 4-10 years) were exposed to TCDD with a total dose of 40.5-42.0 or 405-420ng/kg bodyweight by repeated subcutaneous injections starting at gestational day 20 and followed by injections every 30 days until 90 days after delivery. At a mean age of 7 years the offspring were sacrificed and the femur bone dissected. Results from peripheral Quantitative Computed Tomography (pQCT) analyses of the metaphyseal part of the femur bones in female offspring showed significant increases in trabecular bone mineral content (BMC; +84.6%, p<0.05, F-value (F)=5.9) in the low-dose treatment group compared with the controls. In the same animals, analysis of the mid-diaphyseal part revealed increases in total BMC (+21.3%, p<0.05, F=5.2) and cortical cross-sectional area (CSA; +16.4%, p<0.01, F=7.4) compared with the controls. In males, changes in biomechanical properties indicating more fragile bone were observed. Displacement at failure were significantly increased in the male low-dose group compared to the controls (+38.0%, p<0.05, F=11). The high dose of TCDD did not induce any significant changes in bone morphology. In conclusion, in utero and lactational low-dose, but not high-dose exposure to 2,3,7,8-TCDD induced disruption of bone tissue development in rhesus monkey, a result suggesting that similar effects might occur in humans also.

Perinatal and postnatal exposure to 4-tert-octylphenol inhibits cortical bone growth in width at the diaphysis in female mice.

The aim of this study was to investigate the effects of 4-tert-octylphenol (OP) on bone growth in vivo. Pregnant mice were exposed to drinking water containing either 1microg/ml OP (LD group) or 10microg/ml OP (HD group) from gestational day 10 and throughout the lactation period. After weaning, the pups were allowed free access to drinking water containing the appropriate OP concentrations. The serum osteocalcin level of the females, but not the males, was significantly lower in the LD and HD groups than in the control group on postnatal day 31. The femurs of the females were analyzed by peripheral quantitative computed tomography and immunohistochemistry for alkaline phosphatase (ALP) was performed in the sections of the formalin-fixed femurs. The periosteal and endosteal circumferences of the cortical bone at the diaphysis were significantly smaller in the LD group, but not the HD group, than in the control group (4% and 6% smaller, respectively), while there were no differences in the cortical bone density, cortical bone area, or cortical thickness among the three groups. There were fewer ALP-positive cells on the periosteal surfaces at the diaphysis in the LD group than in the control group. The values of the strength strain index (xSSI, ySSI, and pSSI) decreased with decreasing the periosteal circumference. In conclusion, the exposure of female mice to OP during the perinatal and postnatal periods inhibited the periosteal bone formation in the cortical bone at the diaphysis of the femur, thereby causing a reduction in bone growth in width.

[Changes of the femoral bone microstructure in the reindeer during the fetal period of ontogenesis].

The diaphysis of the femoral bone was studied morphometrically in 30 reindeer fetuses aged 2-7.5 months. The formation of the diaphyseal cavity of the femoral bone took place from 3 till 7.5 months, rapidly progressing during all the fetal period. The thickness of the periosteum in the epiphysis and the diaphysis of the bone increased from 2 to 6 months inclusive, whereupon it decreased by the time of birth. The cartilaginous tissue in the epiphyses was present from 2 till 7.5 months; its growth was registered up to 3 months in a proximal epiphysis, while it continued till 4 months in a distal epiphysis. Later on, the thinning of the cartilage was noted till the birth. The thickness of spongy substance of bone epiphyses increased with the fetal age. The osteons in the diaphysis of the femoral bone were formed in 2-month-old fetus, their numbers were found to increase with age. The compact substance of the diaphysis of the femoral bone increased in thickness till 5 months, whereupon the process of thinning of tissue till the time of birth, was noted.

Genetic variation in bone growth patterns defines adult mouse bone fragility.

UNLABELLED: Femoral morphology and composition were determined for three inbred mouse strains between ages E18.5 and 1 year. Genotype-specific variation in postnatal, pubertal, and postpubertal growth patterns and mineral accrual explained differences in adult bone trait combinations and thus bone fragility. INTRODUCTION: Fracture risk is strongly regulated by genetic factors. However, this regulation is generally considered complex and polygenic. Therefore, the development of effective genetic-based diagnostic and treatment tools hinges on understanding how multiple genes and multiple cell types interact to create mechanically functional structures. The goal of this study was to connect variability in whole bone mechanical function, including measures of fragility, to variability in the biological processes underlying skeletal development. We accomplished this by testing for variation in bone morphology and composition among three inbred mouse strains from E18.5 to 1 year of age. MATERIALS AND METHODS: Mid-diaphyseal cross-sectional areas, diameters, moments of inertia, and ash content were determined for three strains of mice with widely differing adult whole bone femoral mechanical properties (A/J, C57BL/6J, and C3H/HeJ) at E18.5 and postnatal days 1, 7, 14, 28, 56, 112, 182, and 365 (n = 5-15 mice/strain/age). RESULTS: Significant differences in the magnitude and rate of change in morphological and compositional bone traits were observed among the three strains at each phase of growth, including prenatal, postnatal, pubertal, and adult ages. These genotype-specific variations in growth patterns mathematically determined how variation in adult bone trait combinations and mechanical properties arose. Furthermore, six bone traits were identified that characterize phenotypic variability in femoral growth. These include (1) bone size and shape at postnatal day 1, (2) periosteal and (3) endosteal expansion during early growth, (4) periosteal expansion and (5) endosteal contraction in later growth, and (6) ash content. These results show that genetic variability in adult bone traits arises from variation in biological processes at each phase of growth. CONCLUSIONS: Inbred mice achieve different combinations of adult bone traits through genotype-specific regulation of bone surface activity, growth patterns, and whole bone mineral accrual throughout femoral development. This study provides a systematic approach, which can be applied to the human skeleton, to uncover genetic control mechanisms influencing bone fragility.

Bone morphogenetic protein (BMP) localization in developing human and rat growth plate, metaphysis, epiphysis, and articular cartilage.

We assessed the distribution and relative staining intensity of bone morphogenetic protein (BMP)-1-7 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five 10-week-old Sprague-Dawley rats. In the rats, articular cartilage was also examined. BMP proteins were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-1-7), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-1-6), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-1-7). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-1-7 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of bone remodeling.

Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis.

Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues.

Analysis of shrinkage in human fetal diaphyseal lengths from fresh to dry bone using Petersohn and Köhler's data.

Calculation of gestational age from forensic fetal remains may be problematic. If soft-tissue indicators are not available, then diaphyseal lengths obtained through sonograms on living fetuses in utero or radiographs of long bones can be compared to known European standards, such as Fazekas and Kósa (1978) and Olivier and Pineau (1958, 1960). Radiographic comparison to these European standards, however, requires a correction factor for diaphyseal shrinkage from fresh to dry states. Percent shrinkage is calculated for six diaphyses (humerus, ulna, radius, femur, tibia and fibula) from Petersohn and Köhler's data published in Fazekas and Kósa (1978:362-369). Average shrinkage, standard deviation, minimum and maximum values are calculated for each diaphysis and for all diaphyses during 4-10 lunar months (LM) and newborns. Corresponding average and standard deviation values are as follows: 4 LM-10.09% +/- 2.67%; 5 LM-5.74% +/- 0.84%; 6 LM-3.48% +/- 0.49%; 7 LM-2.32% +/- 0.16%; 8 LM-2.18% +/- 0.51%; 9 LM-1.76% +/- 0.14%; 10 LM-1.90% +/- 0.59%; and newborns-1.28% +/- 0.55%. Analysis of these values suggests that percent shrinkage steadily declines as the fetus ages. This pattern presumably reflects calcification of bone during growth and development in utero. These findings demonstrate a significant shrinkage in the diaphysis early in development, which may alter accurate age estimation in the earliest fetal age groups.

A quantitative study of human fetal femur growth (from the 15th to 24th weeks postconception)

This study aims to analyze the human femur growth. The materials utilized are 140 dissected femures from 70 Brazilian fetuses, age ranging from the 15th to the 24th gestational week. Three different measurements were made: Diaphyseal Length (DL), Total Lenghth (TL) and Diaphyseal Diameter (DD). We correlated these measurements to Gestational Age (GA) and to Crown-Rump Length (CR) and found that diaphysea length is best corelated to both parameters. This indicates diaphyseal lengh as an useful parameter, in the second trimester, for determining gestational age or as additional parameter to monitor fetal growth, helping in the diagnosis of growth's abnormalities.

An ultrastructural study of the perichondrium in cartilages of the chick embryo.

The ultrastructure of perichondrial tissue of cartilage rudiments (metatarsus, tibiotarsus and sternum) of the chick embryo at various stages of development (H.H. stages 28-45) was investigated by transmission electron microscopy. Previous microscopic and submicroscopic data were generally confirmed, but new findings indicated: (a) the existence of a temporary syncytial state of perichondroblasts during the earliest developmental stages, (b) the existence of a perichondrial cambial layer of stem cells, (c) involvement of perichondroblasts in the appositional growth of cartilage. Electron microscopy revealed clear temporal relations between cell differentiation, perichondrial growth and the structure and production of perichondrial ECM. In addition, the boundaries between cartilage and perichondrial tissue were demonstrated unambiguously. Perichondrial structure varied specifically with each cartilage segment; in particular the perichondrium in long bone rudiments (where ossification starts early) contrasted with that in the permanent cartilage medial process of the sternum.

Technical note: error in Olivier and Pineau's regression formulae for calculation of stature and lunar age from radial diaphyseal length in forensic fetal remains.

Stewart ([1979] Essentials of Forensic Anthropology, pp. 128-131) cites five regression formulae presented by Olivier and Pineau ([1960] Ann. Méd. Lég. 40:141-144) for estimation of fetal stature from diaphyseal length. Of these formulae, one appears problematic: the formula for calculation of stature from the radius yields values which suggest the fetus has a much greater crown-heel (CH) length than do the remaining formulae for the diaphyses of other long bones. Moreover, when this stature estimate, so derived, is then inserted into these authors' earlier general formula for estimation of lunar age (Olivier and Pineau [1958] Arch. Anat. 6:21-28) the error is compounded. A fetus is now indicated to be nearly a trimester older than when the CH lengths obtained by the other long bone formulae are used. Accordingly, we believe this particular formula, unlike the others, is incorrect and should not be used to estimate lunar age from fetal remains.