Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days, The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11), The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH, Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy

Objective: This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Materials and methods: Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 mm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Results: Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. Conclusion: These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

High concentration of residual aluminum oxide on titanium surface inhibits extracellular matrix mineralization

In the present study we characterized titanium (Ti) surfaces submitted to different treatments and evaluated the response of osteoblasts derived from human alveolar bone to these surfaces. Five different surfaces were evaluated: ground (G), ground and chemical etched (G1-HF for 60 s), sand blasted (SB-Al2O3 particles 65 pm), sand blasted and chemical etched (SLA1-HF for 60 s and SLA2-HF for 13 s). Surface morphology was evaluated under SEM and roughness parameters by contact scanning instrument. The presence of Al2O3 was detected by EDS and the amount calculated by digital analyses. Osteoblasts, were cultured on these surfaces and it was evaluated: cell adhesion, proliferation, and viability, alkaline phosphatase activity, total protein content, and matrix mineralization formation. Physical and chemical treatments produced very different surface morphologies. Al2O3 residues were detected on SB and SLA2 surfaces. Only matrix mineralization formation was affected by different surface treatments, being increased on rough surface (SLA1) and reduced on surface with high amount of Al2O3 residues (SB). On the basis of these findings, it is possible to conclude that high concentration of residual Al2O3 negatively interfere with the process of matrix mineralization formation in contact with Ti implant surfaces. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 87A: 588-597, 2008

Bril: A novel bone-specific modulator of mineralization

In the course of attempting to define the bone ""secretome"" using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 1.34 arnino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus-mediated Brit overexpression and lentivirus-mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose-dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.

Effects of high-dose etidronate treatment on microdamage accumulation and biomechanical properties in beagle bone before occurrence of spontaneous fractures

We recently demonstrated that suppressed bone remodeling allows microdamage to accumulate and causes reductions in some mechanical properties. However, in our previous study, I year treatment with high-dose etidronate (EHDP) did not increase microdamage accumulation in most skeletal sites of dogs in spite of complete remodeling suppression and the occurrence of spontaneous fractures of ribs and/or thoracic spinous processes. This study evaluates the effects of EHDP on microdamage accumulation and biomechanical properties before fractures occur. Thirty-six female beagles, 1-2 years old, were treated daily for 7 months with subcutaneous injections of saline vehicle (CNT) or EHDP at 0.5 (E-low) or 5 mg/kg per day (E-high). After killing, bone mineral measurement, histomorphometry, microdamage analysis, and biomechanical testing were performed. EHDP treatment suppressed intracortical and trabecular remodeling by 60%-75% at the lower dose, and by 100% at the higher dose. Osteoid accumulation caused by a mineralization deficit occurred only in the E-high group, and this led to a reduction of mineralized bone mass. Microdamage accumulation increased significantly by two- to fivefold in the rib, lumbar vertebra, ilium, and thoracic spinous process in E-low, and by twofold in the lumbar vertebra and ilium in E-high. However, no significant increase in damage accumulation was observed in ribs or thoracic spinous processes in E-high where fractures occur following 12 months of treatment. Mechanical properties of lumbar vertebrae and thoracic spinous processes were reduced significantly in both E-low and E-high. These findings suggest that suppression of bone remodeling by EHDP allows microdamage accumulation, but that osteoid accumulation reduces production of microdamage. (Bone 29:271-278; 2001) (C) 2001 by Elsevier Science Inc. All rights reserved.

Effects of suppressed bone turnover by bisphosphonates on microdamage accumulation and biomechanical properties in clinically relevant skeletal sites in beagles

We recently demonstrated that suppression of bone remodeling allows microdamage to accumulate, leading to reduced bone toughness in the rib cortex of dogs. This study evaluates the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties at clinically relevant skeletal sites in the same dogs. Thirty-six female beagles, 1-2 years old, were divided into three groups. The control group was treated daily for 12 months with saline vehicle (CNT), The remaining two groups were treated daily with risedronate at a dose of 0.5 mg/kg per day (RIS), or alendronate at 1.0 mg/kg per day (ALN) orally, The doses of these bisphosphonates were six times the clinical doses approved for treatment of osteoporosis in humans. After killing, the L-1 vertebra was scanned by dual-energy X-ray absorptiometry (DXA), and the L-2 vertebra and right ilium were assigned to histomorphometry, The L-3 vertebra, left ilium, Th-2 spinous process, and right femoral neck were used for microdamage analysis. The L-4 vertebra and Th-1 spinous process were mechanically tested to failure in compression and shear, respectively. One year treatment with risedronate or alendronate significantly suppressed trabecular remodeling in vertebrae (RIS 90%, ALN 95%) and ilium (RIS 76%, ALN 90%) without impairment of mineralization, and significantly increased microdamage accumulation in all skeletal sites measured. Trabecular bone volume and vertebral strength increased significantly following 12 month treatment. However, normalized toughness of the L-4 vertebra was reduced by 21% in both RIS (p = 0.06) and ALN (p = 0.05) groups. When the two bisphosphonate groups were pooled in a post hoc fashion for analysis, this reduction in toughness reached statistical significance (p = 0.02), This study demonstrates that suppression of trabecular bone turnover by high doses of bisphosphonates is associated with increased vertebral strength, even though there is significant microdamage accumulation and a reduction in the intrinsic energy absorption capacity of trabecular bone. (C) 2001 by Elsevier Science Inc. All rights reserved.

In situ electron paramagnetic resonance (EPR) study of surface oxygen species on Au/ZnO catalyst for low-temperature carbon monoxide oxidation

Some paramagnetic superoxide ions detectable by electron paramagnetic resonance (EPR) can be generated on Au/ZnO catalyst by oxygen adsorption at room temperature as well as at 553 K. In both the cases, the O-2(-) ions are present on the catalyst surface. The disappearance of the O-2(-) signal by the introduction of carbon monoxide over the catalyst surface implies that the O-2(-) ions are either the active oxygen species or the precursors of the active oxygen species. The CO3- species produced are also detected by EPR. (C) 2001 Elsevier Science B.V. All rights reserved.

Nutrient dynamics in Queensland savannas: Implications for the sustainability of land clearing for pasture production

Eucalyptus savannas on low nutrient soils are being extensively cleared in Queensland. In this paper we provide background information relevant to understanding nutrient (particularly nitrogen) dynamics in sub/tropical savanna, and review the available evidence relevant to understanding the potential impact of clearing Eucalyptus savanna on nutrient relations. The limited evidence presently available can be used to argue for the extreme positions that: (i) woody vegetation competes with grasses Cor resources. and tree/shrub clearing improves pasture production, (ii) woody vegetation benefits pasture production. At present, the lack of fundamental knowledge about Australian savanna nutrient relations makes accurate predictions about medium- and long-term effects of clearing on nutrient relations in low nutrient savannas difficult. The future of cleared savannas will differ if herbaceous species maintain all functions that woody vegetation has previously held, or if woody species have functions distinct from those of herbaceous vegetation. Research suggests that savanna soils are susceptible to nitrate leaching, and that trees improve the nutrient status of savanna soils in some situations. The nitrogen capital of cleared savanna is at risk if mobile ions are not captured efficiently by the vegetation. and nitrogen input via N-2 fixation from vegetation and microbiotic crusts is reduced. In order to predict clearing effects on savanna nutrient relations, research should be directed to answering (i) how open or closed nutrient cycles are in natural and cleared savanna, (ii) which functions are performed by savanna constituents such as woody and herbaceous vegetation, native and exotic plant species. termites, and microbiotic 7 crusts in relation to nutrient cycles. In the absence of detailed knowledge about savanna functioning, clearing carries the risk of promoting continuous nutrient depiction.

Nitrification in a Vertisol subsoil and its relationship to the accumulation of ammonium-nitrogen at depth

Unusually high concentrations of ammonium have been observed in a Vertisol below 1 m depth in southeast Queensland. This study investigated the possibility that an absence of nitrification is allowing this ammonium to accumulate and persist over time, and examined the soil environmental characteristics that may be responsible for limiting nitrifying organisms. The possibility that anaerobiosis, soil acidity, soil salinity, low organic carbon concentrations, and/or an absence of active nitrifying microorganisms were responsible for limiting nitrification was examined in laboratory and field studies. The presence/absence of anaerobic conditions was determined qualitatively using a field test to give an indication of electron lability. In addition, an incubation study was conducted and soil environmental conditions were improved for nitrifying organisms by adjusting the pH from 4.4 to 7, adjusting the electrical conductivity from 1.6 to 0.5 dS/m, amending with a soluble carbon substrate at a rate of 500 mg/kg, and using microorganisms from the surface horizon to inoculate to the subsoil. Over a 180-day period no nitrification was detected in the control samples from the incubation study, indicating that an extremely low rate of nitrification is likely to be responsible for allowing ammonium to accumulate in this soil. Analysis of the effect of soil environmental conditions on nitrification revealed that anaerobic conditions did not exist at depth and that pH, EC, organic carbon, and inoculation treatments added in isolation had no effect on nitrification. However, when inoculum was added to the soil in combination with pH, a significant increase in nitrification was observed, and the greatest amount of nitrification was observed when inoculum, pH, and EC treatments were added in combination. It was concluded that the reason for the low rate of nitrification in this soil is primarily the absence of a significant population of active nitrifying microorganisms, which may have been unable to colonise the subsoil environment due to its acidic, and to a lesser extent, its saline environment.

Comparisons between bone and cementum compositions and the possible basis for their layered appearances

In humans, age estimation from the adult skeleton represents an attempt to determine chronological age based on growth and maturational events. In teeth, such events can be characterized by appositional growth layers in midroot cementum. The purpose of this study was to determine the underlying cause of the layered microstructure of human midroot cementum. Whether cementum growth layers are caused by changes in relative mineralization, collagen packing and/or orientation, or by variations in organic matrix apposition was investigated by subjecting midroot sections of human canine teeth to analysis using polarized light and scanning electron microscopy (SEM). Polarized light was used to examine transverse midroot sections in both mineralized and demineralized states. Mineralized sections were also reexamined following subsequent decollagenization. Polarized light was additionally used in the examination of mineralized sections taken transversely, longitudinally, and obliquely from the same tooth root. From the birefringence patterns it was concluded that collagen orientation does not change with varying section plane. Instead, the mineral phase was most responsible for the birefringence of the cementum. SEM studies suggested that neither collagen packing nor collagen orientation change across the width of the cementum, confirming and validating the results of the polarized light examination. Also, SEM analysis using electron backscatter and the electron probe suggested no changes in the mean atomic number density, calcium, phosphate, and sulfur levels across the width of the cementum. Therefore, we conclude that crystalline orientation and/or size is responsible for the layered appearance of cementum. (Bone 30:386-392; 2002) (C) 2002 by Elsevier Science Inc. All rights reserved.

Nitrate in soil humic acids revealed by N-14 nuclear magnetic resonance spectroscopy

Ecosystem management such as plant residue retention and prescribed burning can significantly affect soil organic matter (SOM) composition and, thereby, the closely associated carbon (C) and nitrogen (N) cycling processes, which underpin terrestrial ecosystem productivity and sustainability. Humic acid (HA) is an important SOM component and its chemical composition has attracted much attention. Here we report the first application of N-14 nuclear magnetic resonance (NMR) spectroscopy to soil HA study, revealing the surprising existence of nitrate-N and ammonia-N in the HAs. This newly discovered HA nitrate-N, though in a relatively low concentrations, is closely related to soil N availability and responsive to plant residue management regimes in contrasting forest ecosystems. The HA nitrate-N may be a useful and sensitive biochemical indicator of SOM quality in response to different ecosystem management regimes.

Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cells

Background: Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor Cell proliferation and, following clonal expansion of these cells. promotion of differentiation along the osteogenic lineage. Objectives: We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Method: The cell populations were assessed for mineralization potential after long-term culture in media containing beta-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogcnic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin. osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results: As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP. osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion: The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers.

Immunohistochemical localization of fibromodulin in the periodontium during cementogenesis and root formation in the rat molar

Background: Cementum is essential for periodontal regeneration, as it provides anchorage between the root surface and the periodontal ligament. A variety of macromolecules present in the extracellular matrix of the periodontium, including proteoglycans, are likely to play a regulatory role in cementogenesis. Recently, the small leucine-rich proteoglycan, fibromodulin, has been isolated from bovine periodontal ligament and localized in bovine cementum, as well as in human periodontal ligament. Objective: The aim of this study was to examine the distribution of fibromodulin during cementogenesis and root formation. Methods: A standard indirect immunoperoxidase technique was employed, using an antifibromodulin polyclonal antibody on sections of molar teeth from rats aged 3, 5 and 8 weeks. Results: Immunoreactivity to fibromodulin was evident in the periodontal ligament in all sections. An intense positive stain was observed in the extracellular matrix where the periodontal ligament fibers insert into the alveolar bone and where the Sharpey's fibers insert into the cementum. There was no staining evident in the mineralized cellular and acellular cementum. The intensity of immunoreactivity to the antifibromodulin antibody increased proportionally with increasing tissue maturation. Conclusion: The results from this study suggest that fibromodulin is a significant component of the extracellular matrix in the periodontal ligament during development, and may play a regulatory role in the mineralization process or maintaining homeostasis at the hard-soft tissue interface during cementogenesis.