Fluoride effects on bone formation and mineralization are influenced by genetics.

INTRODUCTION: A variation in bone response to fluoride (F(-)) exposure has been attributed to genetic factors. Increasing fluoride doses (0 ppm, 25 ppm, 50 ppm, 100 ppm) for three inbred mouse strains with different susceptibilities to developing dental enamel fluorosis (A/J, a "susceptible" strain; SWR/J, an "intermediate" strain; 129P3/J, a "resistant" strain) had different effects on their cortical and trabecular bone mechanical properties. In this paper, the structural and material properties of the bone were evaluated to explain the previously observed changes in mechanical properties. MATERIALS AND METHODS: This study assessed the effect of increasing fluoride doses on the bone formation, microarchitecture, mineralization and microhardness of the A/J, SWR/J and 129P3/J mouse strains. Bone microarchitecture was quantified with microcomputed tomography and strut analysis. Bone formation was evaluated by static histomorphometry. Bone mineralization was quantified with backscattered electron (BSE) imaging and powder X-ray diffraction. Microhardness measurements were taken from the vertebral bodies (cortical and trabecular bones) and the cortex of the distal femur. RESULTS: Fluoride treatment had no significant effect on bone microarchitecture for any of the strains. All three strains demonstrated a significant increase in osteoid formation at the largest fluoride dose. Vertebral body trabecular bone BSE imaging revealed significantly decreased mineralization heterogeneity in the SWR/J strain at 50 ppm and 100 ppm F(-). The trabecular and cortical bone mineralization profiles showed a non-significant shift towards higher mineralization with increasing F(-) dose in the three strains. Powder X-ray diffraction showed significantly smaller crystals for the 129P3/J strain, and increased crystal width with increasing F(-) dose for all strains. There was no effect of F(-) on trabecular and cortical bone microhardness. CONCLUSION: Fluoride treatment had no significant effect on bone microarchitecture in these three strains. The increased osteoid formation and decreased mineralization heterogeneity support the theory that F(-) delays mineralization of new bone. The increasing crystal width with increasing F(-) dose confirms earlier results and correlates with most of the decreased mechanical properties. An increase in bone F(-) may affect the mineral-organic interfacial bonding and/or bone matrix proteins, interfering with bone crystal growth inhibition on the crystallite faces as well as bonding between the mineral and organic interface. The smaller bone crystallites of the 129P3/J (resistant) strain may indicate a stronger organic/inorganic interface, reducing crystallite growth rate and increasing interfacial mechanical strength.

Vascular system defects and neuronal apoptosis in mice lacking ras GTPase-activating protein.

The gene encoding p120-rasGAP, a negative regulator of Ras, has been disrupted in mice. This Gap mutation affects the ability of endothelial cells to organize into a highly vascularized network and results in extensive neuronal cell death. Mutati ons in the Gap and Nf1 genes have a synergistic effect, such that embryos homozygous for mutations in both genes show an exacerbated Gap phenotype. Thus rasGAP and neurofibromin act together to regulate Ras activity during embryonic development.

Immunolocalization of the Nuk receptor tyrosine kinase suggests roles in segmental patterning of the brain and axonogenesis.

Neural kinase (Nuk) encodes a murine receptor-like tyrosine kinase belonging to the Eph/Elk/Eck family. Protein localization studies indicate that during early embryogenesis Nuk is confined to the developing nervous system, where it marks segments along the axis of the neural tube in the hindbrain (rhombomeres r2, r3 and r5) and specific morphological bulges of the midbrain and forebrain. Subcellular localization of Nuk indicates that this receptor is concentrated at sites of cell-cell contact, often involving migrating neuronal cells or their extensions. Most notably, high levels of Nuk protein are found within initial axon outgrowths and associated nerve fibers. The axonal localization of Nuk is transient and is not detected after migrations have ceased, suggesting a role for this tyrosine kinase during the early pathfinding and/or fasciculation stages of axonogenesis. The subcellular localization of Nuk, as well as the presence of fibronectin type III and immunoglobulin-like adhesive domains on the extracellular region, suggest this receptor tyrosine kinase may function to regulate specific cell-cell interactions during early development of the murine nervous system.

Calcification of chick vertebral chondrocytes grown in agarose gels: a biochemical and ultrastructural study.

Chick embryo vertebral chondrocytes (CHECOV cells) grown in agarose gels form spherical colonies containing cells of hypertrophic morphology and a metachromatically staining matrix. Biochemical analysis of these cultures resulted in the following findings. (i) Calcification of CHECOV cultures can be induced by addition of Pi (at least 1.9 mM) or beta-glycerol phosphate (BGP). (ii) Alkaline phosphatase activity reaches a maximal value at the time when mineral deposition is initiated. (iii) Added BGP is converted to Pi; maximal production of Pi occurs at the time of maximal alkaline phosphatase activity. (iv) BGP-supplemented cultures produce a degree of calcification that corresponds to the amount of BGP conversion to Pi. It can be concluded that Pi is rate-limiting for the calcification of chondrocyte cultures. BGP promotes calcification of these cultures by acting as a substrate for the alkaline phosphatase-mediated production of inorganic phosphate.

Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum.

We have raised an antiserum, designated alpha Enhb-1, to a portion of the mouse En-2 protein containing the homeodomain. The antiserum detects both the En-1 and En-2 proteins in mouse, chick and Xenopus embryos by Western blot analysis. Using whole-mount immunohistochemistry, combined in some cases with scanning electron microscopy, we have examined the distribution of the proteins in the early embryos of these species. The major features of expression were similar. The initial production of En protein occurred, just before or during the formation of the first somites, in a band of the anterior neural plate in the prospective mid/hindbrain region. Later in development En-1 protein accumulated in the ventral ectoderm of the developing mouse and chick limb buds, indicating that a dorsal-ventral polarity is present as soon as any limb bud swelling is apparent and that, at least in the mouse, this polarity is established independently of the apical ectodermal ridge. In all three species, alpha Enhb-1 bound to a subset of ventro-lateral differentiating neurons in the spinal cord and hindbrain and their pattern of birth in the mouse reflected the division of the hindbrain into rhombomeres. En-1 protein also accumulated in a lateral stripe of dermatome in the mouse and chick, indicating a dorsal-ventral subdivision of this tissue. The results show that En expression is a good marker for pattern formation in a variety of tissues and will be useful in experimental studies designed to characterize further these processes.

The emergence and maturation of the first apatite crystals in an in vitro bone formation system.

Using chick periosteal bone forming cultures we have followed the appearance of the first crystals during de Novo bone formation. The first detectable mineral deposits are crystalline. By x-ray diffraction and dark field imaging we found crystal length to grow from around 100 angstroms on day 4 to (135-160) Angstroms by day 8. The Ca/P ratio rose from 1.46 at day 4 to 1.60 at day 8.

Crystal associated diseases: role of scanning electron microscopy in diagnosis.

As crystals are important etiologic agents for disease, their accurate identification in tissues and body fluids is of utmost importance. This paper surveys the roles of crystals in disease process and outlines current analytical techniques for crystal detection and identification in bone tissues. The value of multiple correlated techniques is demonstrated including scanning electron microscopy, x-ray energy spectroscopy and powder diffraction analysis. The current feasibility of utilizing intermediate voltage scanning transmission analytical electron microscopy to integrate these analytical techniques on the same tissue sample is emphasized.

An ultrastructural study of osteogenesis in chick periosteum in vitro.

We described a chick periosteal osteogenesis model at the light microscopic level and biochemically. To confirm that morphologically identifiable bone is being formed, we performed an ultrastructural study of this model. Samples of freshly dissected periostea, as well as whole calvariae, were fixed in glutaraldehyde. Cultured tissues were subsequently harvested and similarly fixed at 2, 4, and 6 days of culture. Examination of freshly dissected periostea showed that these tissues contained many fibroblast-like cells within a loosely arranged connective tissue. There was no evidence of osteoid or mineral in this tissue. Whole calvarial explants contained well-mineralized bone surrounded by an osteoid seam. At 2 days in culture there was an increase in cellular organization as well as extracellular matrix production. No mineral was detected. By 4 days a non-mineralized osteoid matrix was present, which consisted of densely packed, highly oriented, crossbanded collagen fibrils. Early spherical mineral deposits were seen. The osteoid was surrounded by a multilayer of osteoblast-like cells. The 6-day cultures all contained well-mineralized matrix. The mineral was identified by electron diffraction to be apatite. The data suggest that this in vitro model accurately recapitulates the highly ordered and controlled sequence of events leading to de novo bone formation.