Secondary Hyperparathyroidism in Patients with Endemic Skeletal Fluorosis

Investigation of 20 patients with skeletal fluorosis showed that five had clear evidence of secondary hyperparathyroidism. The hyperactivity of the parathyroid glands in skeletal fluorosis in the presence of decreased solubility of the bone mineral (fluoroapatite) strongly suggests that it is a compensatory attempt to maintain a normal extracellular ionized calcium equilibrium. Further study of the parathyroid glands and of bone lesions in skeletal fluorosis is in progress.     

Mineral content of calcified tissues in cystic fibrosis mice

Although abnormal hard tissue mineralization is a recognized complication of cystic fibrosis (CF), the pathogenesis leading from the defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is poorly understood. We hypothesized that CFTR plays a direct role in the mineralization of bone and teeth and tested the hypothesis using CF mouse models [CFTR(−) mice]. In vivo measurements by dual-emission X-ray absorpitometry (DEXA) indicated that bone mineral density (BMD) was reduced in CF mice as compared to gender-matched littermates. However, no change was evident after correction of BMD for the covariant of body weight. The latter finding was confirmed in isolated femurs and nasal bones by standard dry-ashing and instrumental neutron activation analysis (INAA). INAA of the continuously growing hypsodont incisor teeth from CFTR(−) mice revealed reduced Ca and normal P in the enamel layer—a finding consistent with changes in the deciduous teeth of CF children. Interestingly, enamel fluoride was increased in the CFTR(−) incisors and may associate with abnormal enamel crystallite formation. The iron content of the incisor enamel was reduced, explaining the loss of yellow pigmentation in CFTR(−) incisors. In contrast to the incisors, the mineral content of the slow-growing brachydont molar teeth was not different between CFTR(−) and CFTR(+) mice. It was concluded that CFTR does not play a direct role in the mineralization of bones or brachydont teeth in mice. Functional CFTR is apparently required for normal mineralization of the hypsodont incisors. However, multiple changes in the mineral composition of the CF incisors suggest an indirect role for CFTR, perhaps by maintaining a normal salivary environment for continuous tooth eruption. Preliminary reports published in Pediatric Pulmonology, 14, 253A (1997) and 15, 253A (1998).     

Micro-PIGE determination of fluorine distribution in developing hamster tooth germs

A micro-PIGE (Proton-Induced gamma-ray Emission) technique based on the delayed 5/2+----1/2+ nuclear transition of fluorine (E gamma = 197 keV, t1/2 = 87 ns) emitted after 19F(p,p', gamma)19F reaction was used to detect and study the distribution of fluorine in the developing enamel organ during pre-eruptive stages, i.e., the transitional to early maturation stages of enamel formation in neonatal hamsters administered a single IP dose of sodium fluoride (20 mg NaF/kg body weight). The aforementioned nuclear reaction is unique for fluorine, and therefore detection of gamma-rays emanating from this reaction in a biological specimen implies a positive identification of fluorine at that particular site. Calcium and phosphorus X-rays were also recorded and used as parameters for assessment of the relationship between the degree of mineralization and fluoride incorporation into the enamel organ. The highest fluorine concentration in the enamel organ was recorded in the dentin near the dentin-enamel junction (DEJ). In the enamel, the highest concentration of fluorine was found to be associated with the more mature areas of the enamel near the DEJ, but gradually decreased in the direction of the enamel surface. Fluorine was not detected in the control germs. These results suggest that administration of fluoride in high doses during the pre-eruptive stages of enamel formation leads to incorporation of the ion into the forming dentin and enamel mineral, and that the enamel matrix does not seem to bind fluoride avidly.     

Growth potential in onlay bone grafts: a comparison of vascularized and free calvarial bone and suture bone grafts

Vascularized bone grafts are characterized by a viable cell population with osteogenic potential. These features suggest that continued growth can be anticipated following vascularized membranous bone transfer in a growing craniofacial skeleton. The present paper compares the potential for appositional bone growth in vascularized and free calvarial onlay bone grafts. In seven 8-week-old beagles, growth was assessed by direct caliper measurements of graft dimensions intraoperatively and 16 weeks postoperatively. Vascularized grafts demonstrated a 50 to 60 percent increase in size in all dimensions compared to 10 to 20 percent growth in free grafts (p less than 0.01). Microradiography revealed preservation of calvarial bony architecture and minimal resorption in vascularized grafts, while triple-fluorochrome labeling confirmed subperiosteal appositional bone formation. Free grafts were characterized by significant resorption and a delay in subperiosteal bone formation.     

Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice

Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam(+/-) mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam(-/-) mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam(-/-) mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.     

Procion dyes as matrix markers in growing bone and teeth

Additional and extended data on 2726 subjects from Costa Rica, Honduras, Nicaragua and Panama provide confirming evidence for continuing bone expansion of the second metacarpal at midshaft. As shown in 4924 subjects from eight populations, subperiosteal apposition is more rapid in the female than in the male, and accounts for a net increase of 10% in cross-sectional area from age 25 to age 85. Unlike endosteal resorption, which is precipitous in onset, adult subperiosteal gain is continuous from the third through the ninth decades.     

Invited Review: Pathogenesis of osteoporosis.

Patients with fragility fractures may have abnormalities in bone structural and material properties such as larger or smaller bone size, fewer and thinner trabeculae, thinned and porous cortices, and tissue mineral content that is either too high or too low. Bone models and remodels throughout life; however, with advancing age, less bone is replaced than was resorbed within each remodeling site. Estrogen deficiency at menopause increases remodeling intensity: a greater proportion of bone is remodeled on its endosteal (inner) surface, and within each of the many sites even more bone is lost as more bone is resorbed while less is replaced, accelerating architectural decay. In men, there is no midlife increase in remodeling. Bone loss within each remodeling site proceeds by reduced bone formation, producing trabecular and cortical thinning. Hypogonadism in 20-30% of elderly men contributes to bone loss. In both sexes, calcium malabsorption and secondary hyperparathyroidism increase remodeling: more bone is removed from an ever-diminishing bone mass. As bone is removed from the endosteal envelope, concurrent bone formation on the periosteal (outer) bone surface during aging partly offsets bone loss and increases bone's cross-sectional area. Periosteal apposition is less in women than in men; therefore, women have more net bone loss because they gain less on the periosteal surface, not because they resorb more on the endosteal surface. More women than men experience fractures because their smaller skeleton incurs greater architectural damage and adapts less by periosteal apposition.     

A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects

Ameloblastin (AMBN) is the second most abundant extracellular matrix protein produced by the epithelial cells called ameloblasts and is found mainly in forming dental enamel. Inactivation of its expression by gene knockout results in absence of the enamel layer and its replacement by a thin layer of dysplastic mineralized matrix. The objective of this study was to further characterize the enamel organ and mineralized matrix produced in the AMBN knockout mouse. However, in the course of our study, we unexpectedly found that this mouse is in fact a mutant that does not express the full-length protein but that produces a truncated form of AMBN. Mandibles from wild type and mutant mice were processed for morphological analyses and immunolabeling. Microdissected enamel organs and associated matrix were also prepared for molecular and biochemical analyses. In incisors from mutants, ameloblasts lost their polarized organization and the enamel organ detached from the tooth surface and became disorganized. A thin layer of dysplastic mineralized material was deposited onto dentin, and mineralized masses were present within the enamel organ. These mineralized materials generated lower backscattered electron contrast than normal enamel, and immunocytochemistry with colloidal gold revealed the presence of amelogenin, bone sialoprotein and osteopontin. In addition, the height of the alveolar bone was reduced, and the junctional epithelium lost its integrity. Immunochemical and RT–PCR results revealed that the altered enamel organ in the mutant mice produced a shorter AMBN protein that is translated from truncated RNA missing exons 5 and 6. These results indicate that absence of full-length protein and/or expression of an incomplete protein have direct/indirect effects beyond structuring of mineral during enamel formation, and highlight potential functional regions on the AMBN molecule.     

Transmission FT-IR microspectroscopy of mineral phases in calcified tissues

Fourier-transform infrared microspectroscopy (FT-IRM) was used to study bone mineralization processes in an in vivo model and in enamel in osteogenesis imperfecta. Finally, the ability of FT-IRM to map new bone formed in implanted macroporous calcium phosphate biomaterial from sections was reported for the first time. FTIRM allowed the correlation of the microstructure of bone formation in the in vivo model with modifications in carbonate and phosphate environments of the mineral phases during maturation. FT-IRM analysis on enamel sections revealed changes in the mineral environment of carbonate and phosphate ions and probably in the size of enamel crystals. These modifications contributed to the fragility of enamel in osteogenesis imperfecta. The infrared functional group imaging of a part of implanted biomaterial and the bone ingrowth provided the visualization of chemical modifications occurring in biomaterial implants at 20 μm spatial resolution. The use of FT-IRM, in conjunction with appropriate sampling methods and data analysis should provide further insight into the molecular structure of mineral phases of calcified tissues and help to elucidate mineralization processes, skeletal disorders and properties of the biomaterials used as bone substitute.     

Increased intracortical bone remodeling during lactation in beagle dogs

There are substantial changes in skeletal and mineral metabolism during pregnancy and lactation. The purpose of this study was to determine the changes in intracortical bone remodeling and turnover during lactation in beagle dogs. A femur and rib were obtained from dogs near the end of lactation or soon after weaning and compared with nonlactating controls. Rib cortical bone had much higher bone turnover rates than did femoral diaphyseal cortical bone. The number of single-labeled osteons and the number of resorption spaces were significantly greater during lactation in both the rib and the femur. Additionally, the mineral apposition rate, basic multicellular unit activation frequency, and bone turnover rates were greater in the femoral cortical bone from the lactating dogs than from the controls. These data demonstrate that during lactation, intracortical bone remodeling increases, and this may provide a mechanism for the skeleton to be responsive to the calcium requirements of the mother. In addition, these data may help explain the transient decreases in cortical bone mineral density that are reported to occur during human lactation.     

Prospective trial of oestrogen and calcium in postmenopausal women.

In a prospective trial in 72 postmenopausal women to compare the effects on bone loss of no treatment, treatment with oestrogen, and treatment with calcium the women were followed up for at least two years and examined densitometrically and morphometrically. Women in the untreated control group continued to lose bone during the two years, whereas the oestrogen-treated group lost none. Loss in the calcium-treated group was intermediate. Oestrogen appeared to inhibit endosteal bone resorption and may have stimulated subperiosteal bone apposition.     

Thyroid hormone excess rather than thyrotropin deficiency induces osteoporosis in hyperthyroidism

Thyrotoxicosis is an important but under recognized cause of osteoporosis. Recently, TSH deficiency, rather than thyroid hormone excess, has been suggested as the underlying cause. To investigate the molecular mechanism of osteoporosis in thyroid disease, we characterized the skeleton in mice lacking either thyroid hormone receptor alpha or beta (TRalpha(0/0), TRbeta-/-). Remarkably, in the presence of normal circulating thyroid hormone and TSH concentrations, adult TRalpha(0/0) mice had osteosclerosis accompanied by reduced osteoclastic bone resorption, whereas juveniles had delayed endochondral ossification with reduced bone mineral deposition. By contrast, adult TRbeta-/- mice with elevated TSH and thyroid hormone levels were osteoporotic with evidence of increased bone resorption, whereas juveniles had advanced ossification with increased bone mineral deposition. Analysis of T3 target gene expression revealed skeletal hypothyroidism in TRalpha(0/0) mice, but skeletal thyrotoxicosis in TRbeta-/- mice. These studies demonstrate that bone loss in thyrotoxicosis is independent of circulating TSH levels and mediated predominantly by TRalpha, thus identifying TRalpha as a novel drug target in the prevention and treatment of osteoporosis.     

Developmental and Post-Eruptive Defects in Molar Enamel of Free-Ranging Eastern Grey Kangaroos (Macropus giganteus) Exposed to High Environmental Levels of Fluoride

Dental fluorosis has recently been diagnosed in wild marsupials inhabiting a high-fluoride area in Victoria, Australia. Information on the histopathology of fluorotic marsupial enamel has thus far not been available. This study analyzed the developmental and post-eruptive defects in fluorotic molar enamel of eastern grey kangaroos (Macropus giganteus) from the same high-fluoride area using light microscopy and backscattered electron imaging in the scanning electron microscope. The fluorotic enamel exhibited a brownish to blackish discolouration due to post-eruptive infiltration of stains from the oral cavity and was less resistant to wear than normally mineralized enamel of kangaroos from low-fluoride areas. Developmental defects of enamel included enamel hypoplasia and a pronounced hypomineralization of the outer (sub-surface) enamel underneath a thin rim of well-mineralized surface enamel. While the hypoplastic defects denote a disturbance of ameloblast function during the secretory stage of amelogenesis, the hypomineralization is attributed to an impairment of enamel maturation. In addition to hypoplastic defects, the fluorotic molars also exhibited numerous post-eruptive enamel defects due to the flaking-off of portions of the outer, hypomineralized enamel layer during mastication. The macroscopic and histopathological lesions in fluorotic enamel of M. giganteus match those previously described for placental mammals. It is therefore concluded that there exist no principal differences in the pathogenic mechanisms of dental fluorosis between marsupial and placental mammals. The regular occurrence of hypomineralized, opaque outer enamel in the teeth of M. giganteus and other macropodids must be considered in the differential diagnosis of dental fluorosis in these species.     

Population frequencies and altered remodeling mechanisms in normal medullary stenosis

As shown in 2065 women aged 18 to 45 years from seven countries of Central and North America, the population frequency of normal medullary stenosis ranges from under 2% to nearly 7%. In this condition, endosteal apposition may begin in infancy and continue through the fourth decade with a compensatory reduction in the rate of subperiosteal apposition, leading to an approximately normal cortical cross-sectional area contained in a smaller subperiosteal envelope.     

Time course for bone formation with long-term external mechanical loading.

Increased mechanical loading of bone with the rat tibia four-point bending device stimulates bone formation on periosteal and endocortical surfaces. With long-term loading cell activity diminishes, and it has been reported that early gains in bone size may reverse. This study examined the time course for bone cellular and structural response after 6, 12, and 18 wk of loading at 1,200-1, 700 microstrain (muepsilon). Bone formation rates, measured by histomorphometry, were compared within groups, between loaded and contralateral nonloaded tibiae, and between weeks. Formation surface, mineral apposition rate, and bone formation rate on periosteal and endocortical surfaces were elevated after 6 wk of loading. By 12 wk of loading, periosteal and endocortical formation surface and endocortical mineral apposition rates were elevated. By 18 wk of loading, periosteal adaptation appeared complete, whereas endocortical mineral apposition rate remained elevated. No periosteal resorption was observed. Average thickness of new bone formed, from baseline to collection, was greater in loaded than nonloaded tibiae by week 6 and was maintained through week 18. Early increases in bone formation result in periosteal apposition of new bone that persists after formation ceases.     

Morphological and structural studies of early mineral formation in enamel of rat incisors by electron spectroscopic imaging (ESI) and electron spectroscopic diffraction (ESD)

Morphological and structural analysis of the earliest stage of crystal formation in enamel of rat incisors, by use of energy filtering transmission electron microscopy (EFTEM), has shown needlelike crystallites with a dotlike substructure. We conclude that these dots (nanometer-sized particles) have developed at nucleating, active sites along the non-collagenous matrix proteins in enamel. Calcium and phosphate groups are bound at such active sites and develop to nuclei, which grow to these stable dots (nanometer-sized particles). The dots coalesce rapidly in longitudinal direction, along the matrix proteins, with neighbouring dots to form parallel arranged needlelike crystallites. These needles grow and coalesce in lateral directions to ribbon-platelike crystallites. In enamel most of the organic substance becomes decomposed and transported to the ameloblasts. Consequently, the ribbon-platelike crystallites can coalesce to form much thicker (hydroxy)-apatite crystals than in dentine. Already in the earliest stage of crystal formation the mineral chains of dots (nanometer-sized particles) and the needlelike crystallites show a parallel orientation in the direction of the c-axis of hydroxyapatite. This is supported by the texture of the 002 reflections in the corresponding electron spectroscopic diffraction patterns (ESD), which appear as the first Bragg reflections.     

Fluoride-induced lesions in the teeth of the short-tailed field vole (Microtus agrestis): A description of the dental pathology

The effect of fluoride on the appearance of the teeth of the short-tailed field vole. Microtus agrestis, was investigated in both wild animals collected from field sites affected by different levels of industrial fluoride contamination and laboratory-reared animals consuming experimental grass diets of known fluoride concentration or with known fluoride concentrations in drinking water. The extent and severity of lesions on the surface and structure of both incisors and molars are described as six lesion types and related to the amount of biologically available orally ingested fluoride. In the incisors of voles consuming relatively low fluoride diets, lesions are mainly confined to those resulting from disruption of enamel pigmentation expressing itself as concentric bands of pigmentation-free areas on incisor surfaces. The visible effects on molars at low fluoride levels are confined to minor alterations in surface appearance. At higher levels of available dietary fluoride, effects on enamel pigmentation are superseded by alterations in the formation, composition, and strength of both enamel and dentine. The incisors exhibit a marked to severe increase in the cutting tip erosion rates with comparable increases in the extent of abnormal surface changes (enamel hypoplasia) and loss of enamel pigmentation. The grinding surfaces of molars from animals exposed to high levels of dietary fluoride exhibit increasingly severe erosion of outer enamel regions, combined with cavitation and staining of the exposed central dentine. The mechanisms through which fluoride elicits increasingly visible and pathological alterations to the surface and subsurfaces of rodent teeth are discussed. J Morphol 232: 155–167, 1997. © 1997 Wiley-Liss, Inc.     

Long-term remodeling of vascularized and nonvascularized onlay bone grafts: a macroscopic and microscopic analysis

The present study was performed to compare vascularized and nonvascularized onlay bone grafts to investigate the potential effect of graft-to-recipient bed orientation on long-term bone remodeling and changes in thickness and microarchitectural patterns of remodeling within the bone grafts. In two groups of 10 rabbits each, bone grafts were raised bilaterally from the supraorbital processes and placed subperiosteally on the zygomatic arch. The bone grafts were oriented parallel to the zygomatic arch on one side and perpendicular to the arch on the contralateral side. In the first group, vascularized bone grafts were transferred based on the auricularis anterior muscle, and in the second group nonvascularized bone grafts were transferred. Fluorochrome markers were injected during the last 3 months of animal survival, and animals were killed either 6 or 12 months postoperatively. The nonvascularized augmented zygoma showed no significant change in thickness 6 months after bone graft placement and a significant decrease in thickness 1 year after graft placement (p < 0.01). The vascularized augmented zygoma showed a slight but statistically significant decrease in thickness 6 months after graft placement (p < 0.003), with no significant difference relative to its initial thickness 1 year after graft placement. In animals killed 6 months after bone graft placement, both the rate of remodeling and the bone deposition rate measured during the last 3 months of survival were significantly higher in the vascularized bone grafts compared with their nonvascularized counterparts (p < 0.02). By 1 year postoperatively, there were no significant differences in thickness, mineral apposition rate, or osteon density between bone grafts oriented perpendicular and parallel to the zygomatic arch. These findings indicate that the vascularity of a bone graft has a significant effect on long-term thickness and histomorphometric parameters of bone remodeling, whereas the direction of placement of a subperiosteal graft relative to the recipient bed has minimal effect on these parameters. In vascularized bone grafts, both bone remodeling and deposition are accelerated during the initial period following graft placement. Continued bone deposition renders vascularized grafts better suited for the long-term maintenance of thickness and contour relative to nonvascularized grafts.     

Osteomesopyknosis: A Case Report and Review Of Sclerosing Bone Disorders

ObjectiveWe present a case of osteomesopyknosis, a nonmalignant sclerosing bone dysplasia of the axial skeleton, with unknown etiology and unknown prevalence.MethodsWe studied a 49-year-old female who suffered from back and pelvic pain. Her history was obtained, a physical examination performed, and the laboratory results and imaging diagnostics were studied to describe her disease.ResultsA 49-year-old, perimenopausal female suffered excruciating, intermittent, dull back and pelvic pain for 1.5 years. Nonmalignant blastic bone lesions along her spine and pelvis were discovered on computed tomography (CT) and confirmed by magnetic resonance imaging (MRI) and bone scans. Other metabolic/endocrine conditions were ruled out. Her son also has similar symptoms, and corresponding changes were observed on his vertebrae MRI. Both were diagnosed with osteomesopyknosis.ConclusionOsteomesopyknosis is a rare, autosomal dominant condition characterized by nonmalignant osteosclerosis restricted to the axial skeleton. The disease may produce chronic low-grade back pain in the thoracic and lumbar regions. (Endocr Pract. 2014;20:e106-e111)     

High bone mass gained by exercise in growing male mice is increased by subsequent reduced exercise.

Exercise-induced bone gains are lost if exercise ceases. Therefore, continued exercise at a reduced frequency or intensity may be required to maintain these benefits. In this study, we evaluated whether 4 wk of reduced exercise after 4 wk of running exercise in growing male mice results in the maintenance of high bone mass. Five-week-old mice were divided into the following groups: 1) baseline control; 2) 4-wk control; 3) 4-wk exercise; 4) 8-wk control; 5) 4-wk exercise followed by 4-wk cessation of training; and 6) 4-wk exercise followed by reduced exercise at half the frequency. The regimen consisted of exercise 6 days/wk, and the reduced exercise regimen consisted of running 3 days/wk on a treadmill for 30 min/day, at 12 m/min on a 10 degrees uphill slope. Running exercise significantly increased bone mineral density of the femur, periosteal mineral apposition rate, bone formation rate, percent labeled perimeter at the midfemur, and osteogenic activity of bone marrow cells. However, these parameters declined to the age-matched sedentary control after cessation of training. In contrast, the reduced exercise group had significantly higher mineral apposition rate compared with those of the sedentary control and cessation of training groups. Furthermore, bone mineral density for the reduced exercise group was significantly higher than those for the other groups. These results suggest that the high bone formation gained through exercise can be maintained, and bone mass was further increased by subsequent exercise even if the exercise frequency is reduced.